Multi-dimensional chromatographic methods for separating n-glycans

ABSTRACT

A multi-dimensional chromatographic method for the separation of N-glycans. The method comprises providing a glycan preparation that includes at least one negatively charged N-glycan. The glycan preparation is then separated by anion-exchange chromatography and at least one secondary chromatographic technique.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/923,705, filed Apr. 16, 2007, the entire contents of which areincorporated herein by reference.

BACKGROUND

Sugar-containing biomolecules, such as glycans and glycoconjugates,provide significant challenges in their characterization,quantification, purification, and structure elucidation. Such challengesstem from an inherent conformational complexity and structuraldiversity, as well as other physical features, such as instability toisolation conditions, high or low pH, or elevated temperatures.

These challenges are compounded when the biomolecule is provided in acomplex mixture. Such a scenario demands use of separation techniquesand careful handling of the sample. Most separations involvederivatization of the glycan with a suitable label (e.g., achromophore), separation of a desired derivatized glycan from a mixturevia a separation technique, purification, and so on, followed bystructure determination. In some cases, the above methods may alsoinclude release of the glycan component from a glycoconjugate bychemical or enzymatic cleavage prior to derivatization and separation.While methods for separating glycan mixtures have been described in theart there remains a need for other methods.

SUMMARY

The present disclosure provides a multi-dimensional chromatographicmethod for the separation of N-glycans. The method comprises providing aglycan preparation that includes at least one negatively chargedN-glycan. The glycan preparation is then separated by anion-exchangechromatography and at least one secondary chromatographic technique.

Two-dimensional and three-dimensional mapping techniques of N-linkedoligosaccharides have been described by Takahashi and co-workers (see,for example, Takahashi et al., Analytical Biochemistry (1993)208:96-109; Nakagawa et al., Eur. J. Biochem. (1996) 237:76-85;Takahashi et al., Glycoconjugate Journal (1998) 15:905-914; Tomiya andTakahashi, Analytical Biochemistry (1998) 264:204-210; Takahashi et al.,Glycoconjugate Journal (1999) 16:405-414; Takahashi et al., Eur. J.Biochem. (2003) 270:2627-2632; and Yagi et al., Glycobiology (2005)15:1051-1060). This mapping involves chromatographic analysis of a largeselection of known oligosaccharides using fixed standard parameters(e.g., the same column types, the same elution rates and same elutantetc.) in order to provide a comprehensive “map” of their elutionpositions. The relative structure of unknown N-linked oligosaccharidescan be estimated by comparing the elution position (expressed in glucoseunits) of an unknown sample with those of the standard oligosaccharides.

By contrast, the present disclosure provides flexible (i.e., not fixed)procedures that can be tailored for the separation of individualN-glycans from glycan preparations. Among other things, the presentdisclosure demonstrates the particular utility of an anion exchangecolumn, followed by a second separation, in order to isolate and/oranalyze N-linked glycans. These techniques do not employ fixed standardparameters. Rather, the methods can be varied, for example, depending onthe glycan being separated and/or on the source of the initial glycanprepartion. For example, upon separation of a particular glycanpreparation in the first dimension, individual fractions are obtainedwhich comprise one or more N-glycans. Second dimensional parameters(e.g., column type, elution rate, elutant) can then be tuned to eachindividual fraction in order to optimize separation of individualN-glycans from that fraction. In general, the inventors have found that,depending upon the glycan preparation, good resolution of earlierfractions (e.g., such as the first half to two-thirds of the fractions)can be obtained using a normal phase column in the second dimension, andgood resolution of later fractions (e.g., such as the last half toone-third of the fractions) can be obtained using reverse phase columnin the second dimension. In fact, the inventors have surprisingly foundthat a reverse phase porous graphatized carbon (PGC) often effectsbetter separation of later fractions than does a normal phase amidecolumn.

Depending upon the quality of separation in the second dimension,additional separations using additional dimensions may be employed.Thus, each dimension may employ different parameters, and within eachsecond, third, fourth, fifth, etc. dimension, separation parameters canvary from fraction to fraction.

DEFINITIONS

Approximately, About: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterms “approximately” or “about” refer to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value.

Biological sample: The term “biological sample”, as used herein, refersto any solid or fluid sample obtained from, excreted by or secreted byany living cell or organism, including, but not limited to, tissueculture, bioreactors, human or animal tissue, plants, fruits,vegetables, single-celled microorganisms (such as bacteria and yeasts)and multicellular organisms. For example, a biological sample can be abiological fluid obtained from, e.g., blood, plasma, serum, urine, bile,seminal fluid, cerebrospinal fluid, aqueous or vitreous humor, or anybodily secretion, a transudate, an exudate (e.g., fluid obtained from anabscess or any other site of infection or inflammation), or fluidobtained from a joint (e.g., a normal joint or a joint affected bydisease such as a rheumatoid arthritis, osteoarthritis, gout or septicarthritis). A biological sample can also be, e.g., a sample obtainedfrom any organ or tissue (including a biopsy or autopsy specimen), cancomprise cells (whether primary cells or cultured cells), mediumconditioned by any cell, tissue or organ, tissue culture.

Cell-surface glycoprotein: As used herein, the term “cell-surfaceglycoprotein” refers to a glycoprotein, at least a portion of which ispresent on the exterior surface of a cell. In some embodiments, acell-surface glycoprotein is a protein that is positioned on the cellsurface such that at least one of the glycan structures is present onthe exterior surface of the cell.

Cell-surface glycan: A “cell-surface glycan” is a glycan that is presenton the exterior surface of a cell. In many embodiments of thedisclosure, a cell-surface glycan is covalently linked to a polypeptideas part of a cell-surface glycoprotein. A cell-surface glycan can alsobe linked to a cell membrane lipid.

Glycan: As is known in the art and used herein “glycans” are sugars.Glycans can be monomers or polymers of sugar residues, but typicallycontain at least three sugars, and can be linear or branched. A glycanmay include natural sugar residues (e.g., glucose, N-acetylglucosamine,N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose,ribose, xylose, etc.) and/or modified sugars (e.g., 2′-fluororibose,2′-deoxyribose, phosphomannose, 6′ sulfon-acetylglucosamine, etc). Theterm “glycan” includes homo and heteropolymers of sugar residues. Theterm “glycan” also encompasses a glycan component of a glycoconjugate(e.g., of a glycoprotein, glycolipid, proteoglycan, etc.). The term alsoencompasses free glycans, including glycans that have been cleaved orotherwise released from a glycoconjugate.

Glycan preparation: The term “glycan preparation” as used herein refersto a set of glycans obtained according to a particular productionmethod. In some embodiments, glycan preparation refers to a set ofglycans obtained from a glycoprotein preparation (see definition ofglycoprotein preparation below).

Glycoconjugate: The term “glycoconjugate”, as used herein, encompassesall molecules in which at least one sugar moiety is covalently linked toat least one other moiety. The term specifically encompasses allbiomolecules with covalently attached sugar moieties, including forexample N-linked glycoproteins, O-linked glycoproteins, glycolipids,proteoglycans, etc.

Glycoform: The term “glycoform”, is used herein to refer to a particularform of a glycoconjugate. That is, when the same backbone moiety (e.g.,polypeptide, lipid, etc.) that is part of a glycoconjugate has thepotential to be linked to different glycans or sets of glycans, theneach different version of the glycoconjugate (i.e., where the backboneis linked to a particular set of glycans) is referred to as a“glycoform”.

Glycolipid: The term “glycolipid” as used herein refers to a lipid thatcontains one or more covalently linked sugar moieties (i.e., glycans).The sugar moiety(ies) may be in the form of monosaccharides,disaccharides, oligosaccharides, and/or polysaccharides. The sugarmoiety(ies) may comprise a single unbranched chain of sugar residues ormay be comprised of one or more branched chains. In certain embodimentsof the disclosure, sugar moieties may include sulfate and/or phosphategroups. In certain embodiments, glycoproteins contain O-linked sugarmoieties; in certain embodiments, glycoproteins contain N-linked sugarmoieties.

Glycoprotein: As used herein, the term “glycoprotein” refers to aprotein that contains a peptide backbone covalently linked to one ormore₌sugar moieties (i.e., glycans). As is understood by those skilledin the art, the peptide backbone typically comprises a linear chain ofamino acid residues. In certain embodiments, the peptide backbone spansthe cell membrane, such that it comprises a transmembrane portion and anextracellular portion. In certain embodiments, a peptide backbone of aglycoprotein that spans the cell membrane comprises an intracellularportion, a transmembrane portion, and an extracellular portion. Incertain embodiments, methods of the present disclosure comprise cleavinga cell surface glycoprotein with a protease to liberate theextracellular portion of the glycoprotein, or a portion thereof, whereinsuch exposure does not substantially rupture the cell membrane. Thesugar moiety(ies) may be in the form of monosaccharides, disaccharides,oligosaccharides, and/or polysaccharides. The sugar moiety(ies) maycomprise a single unbranched chain of sugar residues or may comprise oneor more branched chains. In certain embodiments of the disclosure, sugarmoieties may include sulfate and/or phosphate groups. Alternatively oradditionally, sugar moieties may include acetyl, glycolyl, propyl orother alkyl modifications. In certain embodiments, glycoproteins containO-linked sugar moieties; in certain embodiments, glycoproteins containN-linked sugar moieties. In certain embodiments, methods disclosedherein comprise a step of analyzing any or all of cell surfaceglycoproteins, liberated fragments (e.g., glycopeptides) of cell surfaceglycoproteins, cell surface glycans attached to cell surfaceglycoproteins, peptide backbones of cell surface glycoproteins,fragments of such glycoproteins, glycans and/or peptide backbones, andcombinations thereof.

Glycosidase: The term “glycosidase” as used herein refers to an agentthat cleaves a covalent bond between sequential sugars in a glycan orbetween the sugar and the backbone moiety (e.g. between sugar andpeptide backbone of glycoprotein). In some embodiments, a glycosidase isan enzyme. In certain embodiments, a glycosidase is a protein (e.g., aprotein enzyme) comprising one or more polypeptide chains. In certainembodiments, a glycosidase is a chemical cleavage agent.

Glycosylation pattern: As used herein, the term “glycosylation pattern”refers to the set of glycan structures present on a particular sample.For example, a particular glycoconjugate (e.g., glycoprotein) or set ofglycoconjugates (e.g., set of glycoproteins) will have a glycosylationpattern. In some embodiments, reference is made to the glycosylationpattern of cell surface glycans. A glycosylation pattern can becharacterized by, for example, the identities of glycans, amounts(absolute or relative) of individual glycans or glycans of particulartypes, degree of occupancy of glycosylation sites, etc., or combinationsof such parameters.

Glycoprotein preparation: A “glycoprotein preparation”, as that term isused herein, refers to a set of individual glycoprotein molecules, eachof which comprises a polypeptide having a particular amino acid sequence(which amino acid sequence includes at least one glycosylation site) andat least one glycan covalently attached to the at least oneglycosylation site. Individual molecules of a particular glycoproteinwithin a glycoprotein preparation typically have identical amino acidsequences but may differ in the occupancy of the at least oneglycosylation sites and/or in the identity of the glycans linked to theat least one glycosylation sites. That is, a glycoprotein preparationmay contain only a single glycoform of a particular glycoprotein, butmore typically contains a plurality of glycoforms. Differentpreparations of the same glycoprotein may differ in the identity ofglycoforms present (e.g., a glycoform that is present in one preparationmay be absent from another) and/or in the relative amounts of differentglycoforms.

N-glycan: The term “N-glycan”, as used herein, refers to a polymer ofsugars that has been released from a glyconjugate but was formerlylinked to the glycoconjugate via a nitrogen linkage (see definition ofN-linked glycan below).

N-linked glycans: N-linked glycans are glycans that are linked to aglycoconjugate via a nitrogen linkage. A diverse assortment of N-linkedglycans exist, but are typically based on the common corepentasaccharide (Man)₃(GlcNAc)(GlcNAc).

O-linked glycans: O-linked glycans are glycans that are linked to aglyconconjugate via an oxygen linkage. O-linked glycans are typicallyattached to glycoproteins via N-acetyl-D-galactosamine (GalNAc) or viaN-acetyl-D-glucosamine (GlcNAc) to the hydroxyl group of L-serine (Ser)or L-threonine (Thr). Some O-linked glycans also have modifications suchas acetylation and sulfation. In some instances O-linked glycans areattached to glycoproteins via fucose or mannose to the hydroxyl group ofL-serine (Ser) or L-threonine (Thr).

Protease: The term “protease” as used herein refers to an agent thatcleaves a peptide bond between sequential amino acids in a polypeptidechain. In some embodiments, a protease is an enzyme (i.e., a proteolyticenzyme). In certain embodiments, a protease is a protein (e.g., aprotein enzyme) comprising one or more polypeptide chains. In certainembodiments, a protease is a chemical cleavage agent.

Protein: In general, a “protein” is a polypeptide (i.e., a string of atleast two amino acids linked to one another by peptide bonds). Proteinsmay include moieties other than amino acids (e.g., may be glycoproteins)and/or may be otherwise processed or modified. Those of ordinary skillin the art will appreciate that a “protein” can be a completepolypeptide chain as produced by a cell (with or without a signalsequence), or can be a functional portion thereof. Those of ordinaryskill will further appreciate that a protein can sometimes include morethan one polypeptide chain, for example linked by one or more disulfidebonds or associated by other means.

Resin: As used herein, a “resin” is an organic polymer. The polymer maybe naturally occurring or synthetic.

Secondary chromatographic technique: As used herein, a “secondarychromatographic technique” refers to a chromatographic technique whichis used to further separate at least a portion of the product from afirst separation. In one embodiment, the secondary chromatographictechnique is different from the one that was used to peform the firstseparation. The primary and secondary chromatographic techniques maydiffer in kind (e.g., anion-exchange chromatography vs. affinitychromatography) or degree (e.g., two anion-exchange chromatographictechniques that use different elution buffers).

Sialic acid: The term “sialic acid”, as used herein, is a generic termfor the N- or O-substituted derivatives of neuraminic acid, anine-carbon monosaccharide. The amino group of neuraminic acid typicallybears either an acetyl or a glycolyl group in a sialic acid. Thehydroxyl substituents present on the sialic acid may be modified byacetylization, methylation, sulfation, and phosphorylation. Thepredominant sialic acid is N-acetylneuraminic acid (Neu5Ac). Sialicacids impart a negative charge to glycans, because the carboxyl grouptends to dissociate a proton at physiological pH. Exemplary deprotonatedsialic acids are as follows:

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Representative chromatogram depicting separation of N-glycans bycharge via anion exchange chromatography.

FIG. 2. Common core structure of an N-glycan/N-linked glycan.

FIGS. 3A-3U. Exemplary N-glycans.

FIGS. 4A-4J. Representative chromatograms depicting separation ofN-glycan fractions obtained after anion-exchange chromatography bynormal-phase amide chromatography relative to a labeled standard (leftpeak).

FIGS. 5A-5G. Exemplary chromatograms depicting separation of N-glycanfractions (e.g., fraction 3, 5 and 7 of a sample) obtained fromanion-exchange chromatography by tuning the gradient and column type ofthe second dimension to the content of each particular fraction. Forexample, a more shallow second dimension (30-48%) gradient on a normalphase amide column effected better separation of fractions 3 and 5 thana steeper (35-53%) gradient (compare A with B and C with D). Betterseparation was found using the porous graphitized carbon (PGC) columnversus a normal phase amide column for fraction 7 (compare E with F andG).

FIG. 6. Chromatographic resolution of glycans by a second dimensionalamide column method.

FIG. 7A-7C. Chromatographic resolution of glycans by a seconddimensional PGC column method.

FIG. 8. Amide chromatogram for fraction 8 glycans derived via a priorAIEX (anion-exchange) chromatography.

FIG. 9. PGC chromatogram for fraction 8 glycans derived via a prior AIEX(anion-exchange) chromatography. The PGC method gave surprisingly betterseparation than the amide method (FIG. 8) for fractions 7, 8 and 9.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Owing to the complexity of typical glycan pools, we have found that theemployment of a single, one-dimensional, separation technique (even athigh resolution) may not provide the best resolution of individualN-glycan components. The present disclosure provides multi-dimensionalchromatographic methods for the separation of N-glycans. In certainembodiments, this method is a two-dimensional chromatographic method. Incertain embodiments, this method involves more than two separationdimensions. In some aspects, N-glycans are quantified (e.g., in thesecond dimension) relative to a standard (e.g., a labeled standard).

In one aspect, the disclosure provides multi-dimensional chromatographicmethods for the separation of N-glycans, comprising the steps of: (i)providing a glycan preparation, wherein the glycan preparation includesat least one negatively charged N-glycan; and (ii) separating the glycanpreparation by anion-exchange chromatography and at least one secondarychromatographic technique.

In certain embodiments, the multi-dimensional chromatographic methodeffects separation of isomeric N-glycans.

It will be appreciated that the anion-exchange chromatography separationmay be performed at any stage during the multi-dimensionalchromatographic method. In one embodiment, anion-exchange chromatographyis used to perform the initial separation of the glycan preparation(i.e., first dimension). In another embodiment, a different type ofchromatography (e.g., any of those discussed herein) can be used for theinitial separation of the glycan preparation and anion-exchangechromatography can be used to perform a subsequent secondary separation(e.g., second dimension).

In one aspect, a known quantity of a reference N-glycan can be includedin a glycan preparation that is to be separated according to the methodsthat are described herein. The reference N-glycan can then be used toprovide a relative quantification for other glycans in the preparation.

In one embodiment, the reference N-glycan is selected so that it isunlikely to occur naturally in the glycan preparation. This will ensurethat the reference N-glycan does not interfere with the analysis.Alternatively, the reference N-glycan can be labeled with a unique labelthat allows it to be differentiated from other glycans in the glycanpreparation that may be labeled with a different label or a collectionof different labels. The addition of a known quantity of referenceN-glycan to the glycan preparation enables each component of the glycanpreparation to be quantified.

Absolute quantitation of N-glycans can be accomplished by spiking themixture to be analyzed with an appropriate fluorescently-labeledstandard. Relative quantitation of N-glycans can be accomplished bycomparison of fluorescence peak areas of the species that are resolvedby chromatography.

Thus, in certain embodiments, the present disclosure provides a methodof characterizing a mixture of N-glycans, said method comprising stepsof:

(i) providing a glycan preparation, wherein the glycan preparationincludes at least one negatively charged N-glycan and a known quantityof a reference N-glycan, wherein the reference N-glycan is labeled witha labeling agent;

(ii) separating the glycan preparation by anion-exchange chromatographyand at least one secondary chromatographic technique; and

(iii) quantifying at least one N-glycan in the glycan preparationrelative to the reference N-glycan.

Anion-Exchange Chromatography (AIEC)

As discussed above, separation of a glycan preparation is provided inone dimension by anion exchange chromatography (AIEC). In brief, anionexchange chromatography is a chromatographic technique which relies oncharge-charge interactions between a negatively charged compound and apositively charged resin.

Exemplary anion exchange resins (i.e., the stationary phase) include,but are not limited to, quaternary amine resins or “Q-resins” (e.g.,Q-Sepharose®, QAE Sephadex®); diethylaminoethane (DEAE) resins (e.g.,DEAE-Trisacryl®, DEAE Sepharose®, benzoylated naphthoylated DEAE,diethylaminoethyl Sephacel®); Amberjet® resins; Amberlyst® resins;Amberlite® resins (e.g., Amberlite® IRA-67, Amberlite® strongly basic,Amberlite® weakly basic), cholestyramine resin, ProPac® resins (e.g.,ProPac® SAX-10, ProPac° WAX-10, ProPac® WCX-10); TSK-GEL® resins (e.g.,TSKgel DEAE-NPR; TSKgel DEAE-5PW); and Acclaim® resins. In certainembodiments, the anion exchange resin is a Q resin. In certainembodiments, the anion exchange resin is a DEAE resin. In certainembodiments, the DEAE resin is a TSK-GEL® DEAE resin.

Typical mobile phases for anionic exchange chromatography includerelatively polar solutions, such as water and polar organic solvents(e.g., acetonitrile and organic alcohols such as methanol, ethanol, andisopropanol). Thus, in certain embodiments, the mobile phase comprisesabout 0%, 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orabout 100% actetonitrile. In certain embodiments, the mobile phasecomprises between about 1% to about 100%, about 5% to about 95%, about10% to about 90%, about 20% to about 80%, about 30% to about 70%, orabout 40% to about 60% acetonitrile at any given time during the courseof the separation.

In certain embodiments, the mobile phase is buffered. In certainembodiments, the mobile phase is not buffered. In certain embodiments,the mobile phase is buffered to a pH between about 7 to about 14. Incertain embodiments, the mobile phase is buffered to a pH between about7 to about 10. In certain embodiments, the mobile phase is buffered to apH between about 7 to about 8. In certain embodiments, the mobile phaseis buffered to a pH of about 7.

Exemplary buffers for anion exchange chromatography are included inTable 2.

TABLE 2 Buffers for anion exchange chromatography Molecule pKadpKa/degree C. Counter ion Ammonium (NH₄) chloride, bromide iodide,acetate N-methyl piperazine 4.75 −0.015 chloride piperazine 5.68 −0.015chloride or formate L-histidine 5.96 chloride bis-Tris 6.46 −0.017chloride bis-Tris propane 6.80 chloride triethanolamine 7.76 −0.020chloride or acetate Tris 8.06 −0.028 chloride N-methyl-diethanolamine8.52 −0.028 chloride diethanolamine 8.88 −0.025 chloride1,3-diaminopropane 8.64 −0.031 chloride ethanolamine 9.50 −0.029chloride piperazine 9.73 −0.026 chloride 1,3-diaminopropane 10.47 −0.026chloride piperidine 11.12 −0.031 chloride phosphate 12.33 −0.026chloride

In certain embodiments, the buffer is selected from a group consistingof ammonia, ammonium chloride, ammonium acetate, ammonium formate,ammonium phosphate, ammonium carbonate, ammonium bicarbonate, N-methylpiperazine, piperazine, piperadine, L-histidine, Tris, bis-Tris,bis-Tris propane, triethanolamine, N-methyl-diethanolamine,diethanolamine, 1,3-diaminopropane, ethanolamine, and phosphate buffers.In certain embodiments, the buffer is ammonium acetate. In certainembodiments, the buffer is ammonium chloride. In certain embodiments,the buffer is ammonium formate. In certain embodiments, the buffer isammonium phosphate. In certain embodiments, the buffer is ammoniumcarbonate. In certain embodiments, the buffer is ammonium bicarbonate.

In certain embodiments, the temperature of the anion exchange column(which houses the resin) is between about 10° C. and about 50° C. Incertain embodiments, the temperature of the anion exchange column isbetween about 20° C. and about 50° C. In certain embodiments, thetemperature of the anion exchange column is between about 30° C. andabout 50° C. In certain embodiments, the temperature of the anionexchange column is about 40° C.

The column can be maintained at a constant temperature throughout theseparation, e.g., using a commercial column heater. In some embodiments,the column can be maintained at a temperature from about 18° C. to about45° C., e.g., about 18° C., 20° C., 22° C., 25° C., 30° C., 37° C., 40°C. or 45° C. In certain embodiments, for consideration of glycanstability, the column temperature is not set higher than 45° C.

Other Chromatographic Techniques

The multi-chromatographic method of the present disclosure also providesfor separation of the glycan preparation by at least one secondarychromatographic technique in addition to anion exchange chromatography.

In certain embodiments, this step may sequentially employ two, three,four, or more, different secondary chromatographic techniques. Incertain embodiments, the methods employ one secondary chromatographictechnique. In certain embodiments, the methods employ two differentsecondary chromatographic techniques. In certain embodiments, themethods employ three different secondary chromatographic techniques. Incertain embodiments, the methods employ one to three different secondarychromatographic techniques. It is also to be understood that the samechromatographic technique may be used several times during a singleseparation (e.g., with a slightly different column, different elutingconditions, etc.).

Secondary techniques that can be used according to the methods describedherein include, but are not limited to, reverse phase liquidchromatography (RP and RP-HPLC), normal phase liquid chromatography (NPand NP-HPLC), ion-pairing reverse phase chromatography (IP-RP andIPRP-HPLC), size exclusion chromatography, affinity chromatography (ACand AC-HPLC), capillary electrophoresis (CE); fluorophore-assistedcarbohydrate electrophoresis (FACE); electrochromatography, and micellarelectrokinetic chromatography (MEKC). Each of these is described in moredetail below.

In certain embodiments, the secondary chromatographic technique is orincludes reverse phase liquid chromatography. Reverse phase liquidchromatography (RP) is a chromatographic technique which relies ondifferences in polarity between a (non-charged) polar analyte and a(non-charged) non-polar resin. The driving force in the binding of theanalyte to the stationary phase is the decrease in the area of thenon-polar segment of the analyte exposed to the solvent. Thishydrophobic effect is dominated by the decrease in free energy fromentropy associated with the minimization of the ordered analyte-polarsolvent interface. The hydrophobic effect is decreased by adding morenon-polar solvent into the mobile phase. This shifts the partitioncoefficient such that the analyte spends some portion of time movingdown the column in the mobile phase, eventually eluting from the column.The characteristics of the analyte play an important role in itsretention characteristics. In general, an analyte with a longer alkylchain length results in a longer retention time because it increases theanalyte's hydrophobicity. Very large analytes, however, can result inincomplete interaction between the large analyte surface and the alkylchain. Retention time increases with hydrophobic surface area which isroughly inversely proportional to analyte size. Branched chain analyteselute more rapidly than their corresponding isomers because the overallsurface area is decreased.

Stationary phases for reverse phase chromatography include, but are notlimited to, silylated silica (i.e., wherein silica has been treated withRMe₂SiCl, and wherein R is a straight chain alkyl group such as C₁₈H₃₇,C₈H₁₇, or C₄H₇), diphenyl resins, divinylbenzene resins, and carbonresins.

The designations for the RMe₂SiCl reversed phase materials refer to thelength of the hydrocarbon chain. In certain embodiments, reverse phasechromatography may include the use of a C18 reverse phase resin (e.g.,for example, octadecylsilane or octadecylsilica, a.k.a. ODS), C8 reversephase resin, or a C4 reverse phase resin. However, in certainembodiments, use of an ODS column as the secondary chromatographictechnique is specifically excluded.

In certain embodiments, reverse phase chromatography includes the use ofa graphitized-carbon resin (e.g., porous graphitized carbon, PGC)

Typical mobile phases for reverse phase chromatography includerelatively polar solutions, such as water and polar organic solvents(e.g., acetonitrile, organic alcohols), and may or may not include abuffer. In certain embodiments, the reverse phase technique does notinclude a buffer. In certain embodiments, the reverse phase techniquedoes include a buffer. Retention time is increased by the addition ofpolar solvent to the mobile phase and decreased by the addition of morehydrophobic solvent. The retention time is therefore longer for analyteswhich are more non-polar in nature, allowing polar analytes to elutemore readily.

Aside from mobile phase polarity, other mobile phase modifiers canaffect analyte retention. For example, the addition of inorganic saltscauses a linear increase in the surface tension of aqueous solutions,and because the entropy of the analyte-solvent interface is controlledby surface tension, the addition of salts tend to increase the retentiontime. Another important component is pH since this can change thehydrophobicity of the analyte. For this reason most methods use abuffering agent, such as sodium phosphate to control the pH. An organicacid such as formic acid or most commonly trifluoroacetic acid is oftenadded to the mobile phase. These serve multiple purposes by controllingthe pH, neutralizing the charge on any residual exposed silica on thestationary phase and acting as ion pairing agents to neutralize chargeon the analyte. The effect varies depending on use but generallyimproves the chromatography.

In certain embodiments, reverse phase chromatography may include use ofan ion-pair reagent. For example, in ion-pairing reverse phasechromatography (IP-RP) a reverse phase resin is used with a stationaryphase which includes an ion pair reagent (e.g., ion pair of an acid anda base) as an additive. When used with common hydrophobic stationaryphases in the reversed-phase mode, ion pair reagents can be used toselectively increase the retention of charged analytes, and enhance peakshape and retention time when common remedies such as modifying eluentratios or changing stationary phase fail. Exemplary ion-pair reagentsinclude: combinations of acids and bases, such as acetic acid and anorganic amine (e.g., dibutylamine); N-hydroxytetrabutylamine;N-hydroxytriethyldodecylamine; and the like. In other embodiments ofthis method, an ion-pair reagent may be selected from the followinglist: cethexonium bromide, triethylamine, tributylamine, tripentylamine,tetrabutyl ammonium bromide, tetrabutyl ammonium chloride, tetrabutylammonium dihydrogen phosphate, tetrabutyl ammonium hydrogen sulfate,tetrabutyl ammonium hydroxide, tetrabutyl ammonium iodide, tetrabutylphosphonium bromide, tetrabutyl phosphonium hydrogen sulfate, tetradecyltrimethyl ammonium bromide, tetradecyl trimethyl ammoniumhydrogensulfate, tetraethyl ammonium bromide, tetraethyl ammoniumhydrogen sulfate, tetraethyl ammonium hydroxide, tetraheptyl ammoniumbromide, tetrahexylammonium bromide, tetrahexyl ammonium dihydrogenphosphate, tetrahexyl ammonium hydrogen sulfate, tetramethyl ammoniumbromide, tetramethyl ammonium hydrogen sulfate, tetramethyl ammoniumhydroxide, tetramethyl ammonium sulfate, tetraoctyl ammonium bromide,tetrapentyl ammonium bromide, tetrapropyl ammonium bromide, tetrapropylammonium hydrogen sulfate, or tetrapropyl ammonium hydroxide.

In certain embodiments, the secondary chromatographic technique is orincludes normal phase liquid chromatography. Normal phase liquidchromatography (NP) is a chromatographic technique which relies ondifferences in polarity between a (non-charged) non-polar analyte and a(non-charged) polar resin. Polar analytes associate with and areretained by the polar stationary phase. Adsorption strengths increasewith increase in analyte polarity, and the interaction between polaranalytes and the polar stationary phase (relative to the mobile phase)increases the elution time. The interaction strength not only depends onthe functional groups in the analyte, but also on steric factors andstructural isomers are often resolved from one another. Stationaryphases for normal phase chromatography include, but are not limited to,silica gel (silanol), alumina, Fluorisil®, and modified silica gels(e.g., such as cyano-modified silica gel; amine-modified silica gel, andamide-modified silica gel). In certain embodiments, normal phasechromatography includes the use of modified silica gel. In certainembodiments, modified silica gel includes cyano-modified silica gel,amine-modified silica gel, or amide-modified silica gel. In certainembodiments, the amide-modified silica gel is GlycoSep-N.

A typical mobile phase normal phase chromatography includes non-polarorganic solvents such as hydrocarbons (e.g., hexanes, pentanes,cyclohexane), halogenated hydrocarbons (e.g., dichloromethane,chloroform, dichloroethane), aromatic hydrocarbons (e.g., benzene,toluene, xylenes), aromatic halogenated hydrocarbons (e.g.,chlorobenzene), ethers (e.g., tetrahydrofuran, diethylether), esters(e.g., ethyl acetate, isopropylacetate), or mixtures thereof. Organicalcohols (e.g., methanol, ethanol, isopropanol, t-butanol) or otherpolar solvents (e.g., acetonitrile) may be added to the eluting solutionin minor amounts in order to increase overall solvent polarity, anddecrease the retention time of the analytes; more hydrophobic solventstend to increase retention times. Organic bases (e.g., triethylamine,diisopropylethyl amine) may also be used in minor amounts to the mobilephase in order to neutralize the slight acidity of the silica gel, anddecrease the retention time of basic analytes. Water as a component ofthe mobile phase in NP chromatography is excluded.

In certain embodiments, the normal phase chromatography step may beperformed in an aqueous normal phase (ANP) format which encompasses themobile phase region between reversed-phase chromatography (RP) andorganic normal phase chromatography (ONP). Water must be present in themobile phase in order to permit the partitioning of solutes in a “normalphase” order. Mobile phases for ANP are based on an polar organicsolvent (e.g., an organic alcohol, acetonitrile) with a small amount ofwater; thus, the mobile phase is both “aqueous” (water is present) and“normal” (less polar than the stationary phase). Thus, polar analytes(such as acids and amines) are most strongly retained, with retentiondecreasing as the amount of water in the mobile phase increases.Generally, the amount of the nonpolar component in the mobile phase mustbe approximately 50% or greater with the exact point of increasedretention depending on the analyte and the organic component of themobile phase. A true ANP stationary phase will be able to function inboth the reversed phase and normal phase modes with only the amount ofwater in the eluent varying. Thus a continuum of solvents can be usedfrom 100% aqueous to pure organic.

ANP retention has been demonstrated for a variety of polar compounds onthe hydride based stationary phases (see, for example, Pesek andMatyska, Journal of Separation Science (2005) 28:2437-2443; Pesek andMatyska, LCGC (2006) 24:296; Pesek et al., Journal of Separation Science29: 872-880 (2006)). An interesting feature of these phases is that bothpolar and nonpolar analytes can be retained over some range of mobilephase composition (organic/aqueous) as a result of residual silanolgroups acting in a HILIC (hydrophilic interaction chromatography) mode.This property distinguishes it from a pure HILIC column where separationby polar differences is obtained, or a pure RP stationary phase on whichseparation by non-polar differences in analytes is obtained with verylimited secondary mechanisms operating. Another important feature of thehydride-based phases is that for many analyses it is usually notnecessary to use a high pH mobile phase to analyze polar analytes suchas bases. The aqueous component of the mobile phase usually containsfrom 0.1 to 0.5% formic, acetic acid, or trifluoroacetic acid, which iscompatible with detector techniques that include mass spectral analysis.

In certain embodiments, the secondary chromatographic technique is orincludes size exclusion chromatography. Size exclusion chromatography(SEC) also known as gel permeation chromatography or gel filtrationchromatography is a chromatographic technique which separates thecomponents of a mixture on the basis of size. This typically involvespassing the mixture through a material with narrow pores that restrictthe passage of different components to different extents.

In certain embodiments, the secondary chromatographic technique is orincludes affinity chromatography. Affinity chromatography (AC) is achromatographic technique which relies on the property of biologicallyactive substances to form stable, specific, and reversible complexes.The formation of these complexes involves the participation of acombination of common molecular forces such as the van der Waal'sinteractions, electrostatic interactions, dipole-dipole interactions,hydrophobic interactions, and hydrogen bonding. An efficient,biospecific bond is formed by a simultaneous and concerted action ofseveral of these forces in the complementary binding sites. For example,a column with conjugated antibodies or lectins against a particularglycan type can be used to preferentially isolate glycans of that typefrom the remainder of the mixture. In another embodiment, the use of animmobilized metal affinity (IMAC) column can be used, to preferentiallyisolate glycans of a structural subtype which will bind to the IMACresin.

In certain embodiments, the secondary chromatographic technique is orincludes capillary electrophoresis. Capillary electrophoresis (CE) is aseparation technique which separates ionic analytes by their charge andfrictional forces. In traditional electrophoresis, electrically chargedanalytes move in a conductive liquid medium (stationary phase) under theinfluence of an electric field. Separations in a capillaryelectrophoresis system are typically dependent on the analytes havingdifferent electrophoretic mobilities (e.g., separation based on on sizeto charge ratio). However, some classes of analyte cannot be separatedby this effect because they are neutral (uncharged) or because they maynot differ significantly in electrophoretic mobility. Adding asurfactant to the electrolyte can facilitate the separation of neutralcompounds (micellar electrokinetic chromatography, see below). Chargedpolymers can be separated by filling the capillary with a gel matrixthat retards longer strands more than shorter strands (capillary gelelectrophoresis). Some capillary electrophoresis systems can also beused for microscale liquid chromatography or capillaryelectrochromatography.

In certain embodiments, the secondary chromatographic technique is orincludes fluorophore-assisted carbohydrate electrophoresis (FACE) inwhich the glycans are labeled with a fluorophore to faciliate detection(see, for example, Gao and Lehrman, Glycobiology (2003) 13:1 G-3G).Terminal aldehydes of N-glycan residues released by hydrolysis may betagged with charged fluorophores (e.g.,8-aminonaphthalene-1,3,6-trisulfonate (ANTS);8-aminopyrene-1,3,6-trisulfonic acid (APTS); 2-aminobenzoic acid (2AA);3-aminobenzoic acid (3AA); 4-aminobenzoic acid (4AA); etc.) andseparated by electrophoresis (e.g., gel electrophoresis, capillaryelectrophoresis) based on charge and frictional forces. Otherfluorescent labeling agents may be employable by FACE such as, forexample, anthranilic acid (AA); 2-aminopyridine (2AP); 2-aminobenzamide(2AB); 3-aminobenzamide (3AB); 4-aminobenzamide (4AB); 2-aminobenzoicethyl etser (2ABEE); 3-aminobenzoic ethyl etser (3ABEE); 4-aminobenzoicethyl etser (4ABEE); 2-aminobenzonitrile (2ABN); 3-aminobenzonitrile(3ABN); 4-aminobenzonitrile (4ABN); 3-(acetylamino)-6-aminoacridin(AA-AC); 2-aminoacridone (AMAC); methylanthranilate (MA);6-aminoquinoline (6AQ); 2-aminonaphthal ene-1,3,6-trisulfonate (ANT);7-aminomethyl-coumarin (AMC); 2-amino(6-amido-biotinyl)pyridine (BAP);9-fluorenylmethoxy-carbonyl-hydrazide (FMOC-hydrazide);3,5-dimethylanthranilic acid; 2-amino-4,5-dimethoxy-benzoic acid;1,2-diamino-4,5-methylenedioxy-benzene (DMB); and ortho-phenylenediamine(OPD).

In certain embodiments a standard sample (e.g., acid-hydrolyzed dextran)can be run under the same conditions to provide a standard set of bandsthat can be used for alignment purposes.

In certain embodiments, the secondary chromatographic technique is orincludes electrochromatography. Electrochromatography is a combinationof size exclusion chromatography and gel electrophoresis which istraditionally used to resolve and separate large analytes such asproteins. These separation mechanisms operate essentially insuperposition along the length of a gel filtration column to which anaxial electric field gradient has been added. The analytes are separatedby size due to the gel filtration mechanism and by electrophoreticmobility due to the gel electrophoresis mechanism. Additionally thereare secondary chromatographic analyte retention mechanisms.

In certain embodiments, the secondary chromatographic technique is orincludes micellar electrokinetic chromatography. Micellar electrokineticchromatography (MEKC) is a chromatographic technique in which componentsin a mixture are separated by differential partitioning between apseudo-stationary micellar phase and an aqueous mobile phase. In mostapplications, MEKC is performed in open capillaries under alkalineconditions to generate a strong electroosmotic flow. The basic set-upand detection methods used for MEKC are the same as those used incapillary electophoresis that were discussed above. The difference isthat the solution contains a surfactant at a concentration that isgreater than the critical micelle concentration (CMC). Above thisconcentration, surfactant monomers are in equilibrium with micelles.Sodium dodecyl sulfate (SDS) is the most commonly used surfactant inMEKC applications. The anionic character of the sulfate groups of SDScause the surfactant and micelles to have electrophoretic mobility thatis counter to the direction of the strong electroosmotic flow. As aresult, the surfactant monomers and micelles migrate quite slowly,though their net movement is still toward the cathode. During a MEKCseparation, the components of the mixture distribute themselves betweenthe hydrophobic interior of the micelle and hydrophilic buffer solution.

In general, any of these chromatographic techniques may be performed inan HPLC (high performance liquid chromatography) format which is a typeof chromatography in which the eluting solvent is conveyed through thecolumn under high pressure (e.g., approximately 200 psi to 6,000 psi).Chromatographic techniques such as anion exchange chromatography(AIEC-HPLC), reversed phase liquid chromatography (RP-HPLC), normalphase liquid chromatography (NP-HPLC), ion-pairing reverse phasechromatography (IPRP-HPLC), and aqueous normal phase chromatography(ANP-HPLC) may all be performed in an HPLC format. It will also beappreciated that in certain embodiments, any one of thesechromatographic techniques may be performed in an UPLC (ultraperformance liquid chromatography) format in which the eluting solventis conveyed through a column with very small particle sizes (e.g.,between 1.7 microns to 1 micron) under ultra high pressures (e.g.,15,000 psi to 100,000 psi). As can be understood from the abovediscussion, the present disclosure provides, in part, for the use of amulti-dimensional chromatographic method wherein anion-exchangechromatography is used to perform the initial separation of the glycanpreparation (i.e., first dimension) and any of the above-describedsecondary chromatographic methods can be used to perform subsequentsecondary separations (e.g., in the second, third, fourth, fifthdimensions).

For example, in certain embodiments, the method comprises separation ofa glycan preparation by anion exchange chromatography (AIEC) followed byeither reverse phase or normal phase chromatography.

In certain embodiments, the method comprises separation of a glycanpreparation by anion exchange chromatography (AIEC) using a Q resin orDEAE resin followed by either reverse phase or normal phasechromatography.

In certain embodiments, the method comprises separation of a glycanpreparation by anion exchange chromatography (AIEC) followed by HPLCchromatography using amide-modified silica gel (normal phase) or PGC(reverse phase). In certain embodiments, the amide-modified silica gelis GlycoSep-N.

In certain embodiments, the method comprises separation of a glycanpreparation by anion exchange chromatography (AIEC) followed by reversephase chromatography using PGC.

In certain embodiments, the method comprises separation of a glycanpreparation by anion exchange chromatography (AIEC) followed by reversephase chromatography, which is further followed by normal phasechromatography.

For example, in certain embodiments, the method comprises separation ofa glycan preparation by anion exchange chromatography (AIEC) using a Qresin or DEAE resin followed by reverse phase chromatography using PGC,which is further followed by normal phase chromatography usingamide-modified silica gel. In certain embodiments, the amide-modifiedsilica gel is GlycoSep-N.

Applications

It will be appreciated that the techniques described herein can beutilized in any of a variety of applications. In general, thesetechniques are useful in any application that involves the structuralcharacterization of glycans. Techniques of the present disclosure may beparticularly useful in a context that requires the separation of certainglycans in a glycan preparation. It will be appreciated that thetechniques can be used with any sample that includes at least onenegatively charged N-glycan irrespective of the nature of any additionalsample components.

Methods of the present disclosure can be applied to glycan preparationsobtained from a wide variety of sources including, but not limited to,therapeutic formulations and biological samples. A biological sample mayundergo one or more analysis and/or purification steps prior to or afterbeing analyzed according to the present disclosure. To give but a fewexamples, in some embodiments, a biological sample is treated with oneor more proteases and/or glycosidases (e.g., so that glycans arereleased); in some embodiments, glycans in a biological sample arelabeled with one or more detectable markers or other agents that mayfacilitate analysis by, for example, mass spectrometry or NMR. Forpurposes of illustration, examples of such steps are described in moredetail below. Any of a variety of separation and/or isolation steps maybe applied to a biological sample in accordance with the presentdisclosure.

Methods of the present disclosure can be utilized to analyze glycans inany of a variety of states including, for instance, free glycans,glycoconjugates (e.g., glycopeptides, glycolipids, proteoglycans, etc.),or cells or cell components, etc.

Methods of the present disclosure may be used to significantly expediteone or more stages of process development for the production of atherapeutic or other commercially relevant glycoprotein of interest.Non-limiting examples of such process development stages that can beimproved using methods of the present disclosure include cell selection,clonal selection, media optimization, culture conditions, processconditions, and/or purification procedure. Those of ordinary skill inthe art will be aware of other process development stages that can beimproved.

The methods can also be utilized to monitor the extent and/or type ofglycosylation occurring in a particular cell culture, thereby allowingadjustment or possibly termination of the culture in order, for example,to achieve a particular desired glycosylation pattern or to avoiddevelopment of a particular undesired glycosylation pattern.

The methods can also be utilized to assess glycosylation characteristicsof cells or cell lines that are being considered for production of aparticular desired glycoprotein (for example, even before the cells orcell lines have been engineered to produce the glycoprotein, or toproduce the glycoprotein at a commercially relevant level).

In some embodiments of the disclosure, a desired glycosylation patternfor a particular target glycoprotein is known, and the technologydescribed herein allows the monitoring of culture samples to assessprogress of the production along a route known to produce the desiredglycosylation pattern. For example, where the target glycoprotein is atherapeutic glycoprotein, for example having undergone regulatory reviewin one or more countries, it will often be desirable to monitor culturesto assess the likelihood that they will generate a product with aglycosylation pattern as close to identical with the establishedglycosylation pattern of the pharmaceutical product as possible, whetheror not it is being produced by exactly the same route. As used herein,“close to identical” refers to a glycosylation pattern having at least90%, 95%, 98% or 99% correlation to the established glycosylationpattern of the pharmaceutical product. In such embodiments, samples ofthe production culture are typically taken at multiple time points andare compared with an established standard or with a control culture inorder to assess relative glycosylation.

In some embodiments of the present disclosure, a desired glycosylationpattern will be more extensive. For example, in some embodiments, adesired glycosylation pattern shows high (e.g., greater than about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more) occupancy of glycosylationsites; in some embodiments, a desired glycosylation pattern shows, ahigh degree of branching (e.g., greater than about 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more have tri or tetraantennary structures).

In some embodiments of the present disclosure, a desired glycosylationpattern will be less extensive. For example, in some embodiments, adesired glycosylation pattern shows low (e.g., less than about 35%, 30%,25%, 20%, 15% or less) occupancy of glycosylation sites; and/or a lowdegree of branching (e.g., less than about 20%, 15%, 10%, 5%, or lesshave tri or tetraantennary structures).

In some embodiments, a desired glycosylation pattern will be moreextensive in some aspects and less extensive than others. For example,it may be desirable to employ a cell line that tends to produceglycoproteins with long, unbranched oligosaccharide chains.Alternatively, it may be desirable to employ a cell line that tends toproduce glycoproteins with short, highly branched oligosaccharidechains.

In some embodiments, a desired glycosylation pattern will be enrichedfor a particular type of glycan structure. For example, in someembodiments, a desired glycosylation pattern will have low levels (e.g.,less than about 20%, 15%, 10%, 5%, or less) of high mannose or hybridstructures, high (e.g., more than about 60%, 65%, 70%, 75%, 80%, 85%,90% or more) levels of high mannose structures, or high (e.g., more thanabout 60%, 65%, 70%, 75%, 80%, 85%, 90% or more; for example at leastone per glycoprotein) or low (e.g., less than about 20%, 15%, 10%, 5%,or less) levels of phosphorylated high mannose.

In some embodiments, a desired glycosylation pattern will include atleast about one sialic acid. In some embodiments, a desiredglycosylation pattern will include a high (e.g., greater than about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more) level of termini that aresialylated. In some embodiments, a desired glycosylation pattern thatincludes sialylation will show at least about 85%, 90%, 95% or moreN-acetylneuraminic acid and/or less than about 15%, 10%, 5% or lessN-glycolylneuraminic acid.

In some embodiments, a desired glycosylation pattern shows specificityof branch elongation (e.g., greater than about 50%, 55%, 60%, 65%, 70%or more of extension is on α1,6 mannose branches, or greater than about50%, 55%, 60%, 65%, 70% or more of extension is on α1,3 mannosebranches).

In some embodiments, a desired glycosylation pattern will include a low(e.g., less than about 20%, 15%, 10%, 5%, or less) or high (e.g., morethan about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) level ofcore fucosylation.

Whether or not monitoring production of a particular target protein forquality control purposes, the present disclosure may be utilized, forexample, to monitor glycosylation at particular stages of development,or under particular growth conditions.

In some particular embodiments of the present disclosure, the methodscan be used to characterize and/or control or compare the quality oftherapeutic products. To give but one example, the present methodologiescan be used to assess glycosylation in cells producing a therapeuticprotein product. Particularly given that glycosylation can often affectthe activity, bioavailability, or other characteristics of a therapeuticprotein product, methods for assessing cellular glycosylation duringproduction of such a therapeutic protein product are particularlydesirable. Among other things, the methods can facilitate real timeanalysis of glycosylation in production systems for therapeuticproteins.

Representative therapeutic glycoprotein products whose production and/orquality can be monitored in accordance with the present disclosureinclude, for example, any of a variety of hematologic agents (including,for instance, erythropoietins, blood-clotting factors, etc.),interferons, colony stimulating factors, antibodies, enzymes, andhormones.

Representative commercially available glycoprotein products include, forexample:

Protein Product Reference Drug interferon gamma-1b Actimmune ®alteplase; tissue plasminogen activator Activase ®/Cathflo ® Recombinantantihemophilic factor Advate human albumin Albutein ® laronidaseAldurazyme ® interferon alfa-N3, human leukocyte derived Alferon N ®human antihemophilic factor Alphanate ® virus-filtered human coagulationfactor IX AiphaNine ® SD Alefacept; recombinant, dimeric fusion proteinLFA3-Ig Amevive ® bivalirudin Angiomax ® darbepoetin alfa Aranesp ™bevacizumab Avastin ™ interferon beta-1a; recombinant Avonex ®coagulation factor IX BeneFix ™ Interferon beta-1b Betaseron ®Tositumomab Bexxar ® antihemophilic factor Bioclate ™ human growthhormone BioTropin ™ botulinum toxin type A Botox ® alemtuzumab Campath ®acritumomab; technetium-99 labeled CEA-Scan ® alglucerase; modified formof beta-glucocerebrosidase Ceredase ® imiglucerase; recombinant form ofbeta- Cerezyme ® glucocerebrosidase crotalidae polyvalent immune Fab,ovine CroFab ™ digoxin immune Fab, ovine DigiFab ™ rasburicase Elitek ®etanercept Enbrel ® epoietin alfa Epogen ® cetuximab Erbitux ™algasidase beta Fabrazyme ® urofollitropin Fertinex ™ follitropin betaFollistim ™ teriparatide Forteo ® human somatropin GenoTropin ® glucagonGlucaGen ® follitropin alfa Gonal-F ® antihemophilic factor Helixate ®Antihemophilic Factor; Factor XIII Hemofil ® insulin Humalog ®antihemophilic factor/von Willebrand factor complex- Humate-P ® humansomatotropin Humatrope ® adalimumab HUMIRA ™ human insulin Humulin ®recombinant human hyaluronidase Hylenex ™ interferon alfacon-1Infergen ® Eptifibatide Integrilin ™ alpha-interferon Intron A ®palifermin Kepivance anakinra Kineret ™ antihemophilic factor Kogenate ®FS insulin glargine Lantus ® granulocyte macrophage colony-stimulatingfactor Leukine ®/Leukine ® Liquid lutropin alfa, for injection LuverisOspA lipoprotein LYMErix ™ ranibizumab Lucentis ® gemtuzumab ozogamieinMylotarg ™ galsulfase Naglazyme ™ nesiritide Natrecor ® pegfilgrastimNeulasta ™ oprelvekin Neumega ® filgrastim Neupogen ® fanolesomabNeutroSpec ™ (formerly LeuTech ®) somatropin [rDNA]Norditropin ®/Norditropin Nordiflex ® insulin; zinc suspension; NovolinL ® insulin; isophane suspension Novolin N ® insulin, regular; NovolinR ® insulin Novolin ® coagulation factor VIIa NovoSeven ® somatropinNutropin ® immunoglobulin intravenous Octagam ® PEG-L-asparaginaseOncaspar ® abatacept, fully human soluable fusion protein Orencia ™muromomab-CD3 Orthoclone OKT3 ® human chorionic gonadotropin Ovidrel ®peginterferon alfa-2a Pegasys ® pegylated version of interferon alfa-2bPEG-Intron ™ Abarelix (injectable suspension); gonadotropin- Plenaxis ™releasing hormone antagonist epoietin alfa Procrit ® aldesleukinProleukin, IL-2 ® somatrem Protropin ® dornase alfa Pulmozyme ®Efalizumab; selective, reversible T-cell blocker Raptiva ™ combinationof ribavirin and alpha interferon Rebetron ™ Interferon beta 1a Rebif ®antihemophilic factor Recombinate ® rAHF/ntihemophilic factor ReFacto ®lepirudin Refludan ® infliximab Remicade ® abciximab ReoPro ™ reteplaseRetavase ™ rituximab Rituxan ™ interferon alfa-2a Roferon-A ® somatropinSaizen ® synthetic porcine secretin SecreFlo ™ basiliximab Simulect ®eculizumab Soliris ® pegvisomant Somavert ® Palivizumab; recombinantlyproduced, humanized mAb Synagis ™ thyrotropin alfa Thyrogen ®tenecteplase TNKase ™ natalizumab Tysabri ® human immune globulinintravenous 5% and 10% Venoglobulin-S ® solutions interferon alfa-n1,lymphoblastoid Wellferon ® drotrecogin alfa Xigris ™ Omalizumab;recombinant DNA-derived humanized Xolair ® monoclonal antibody targetingimmunoglobulin-E daclizumab Zenapax ® ibritumomab tiuxetan Zevalin ™Somatotropin Zorbtive ™ (Serostim ®)

In some embodiments, the disclosure provides methods in which glycansfrom different sources or samples are compared with one another. Incertain embodiments, the disclosure provides methods used to monitor theextent and/or type of glycosylation occurring in different cellcultures. In some such examples, multiple samples from the same sourceare obtained over time, so that changes in glycosylation patterns (andparticularly in cell surface glycosylation patterns) are monitored. Insome embodiments, one of the samples is a historical sample or a recordof a historical sample. In some embodiments, one of the samples is areference sample. For example, in certain embodiments, methods areprovided herein which can be used to monitor the extent and/or type ofglycosylation occurring in different cell cultures.

In some embodiments, glycans from different cell culture samplesprepared under conditions that differ in one or more selected parameters(e.g., cell type, culture type [e.g., continuous feed versus batch feed,etc.], culture conditions [e.g., type of media, presence orconcentration of particular component of particular medium, osmolarity,pH, temperature, timing or degree of shift in one or more componentssuch as osmolarity, pH, temperature, etc.], culture time isolationsteps, etc.) but are otherwise identical, are compared, so that effectsof the selected parameter(s) on N-glycosylation patterns are determined.In certain embodiments, glycans from different cell culture samplesprepared under conditions that differ in a single selected parameter arecompared so that effect of the single selected parameter on theglycosylation pattern is determined. Among other applications,therefore, use of techniques as described herein may facilitatedetermination of the effects of particular parameters on glycosylationpatterns in cells.

In some embodiments, glycans from different batches of a glycoprotein ofinterest (e.g., a therapeutic glycoprotein), whether prepared by thesame method or by different methods, and whether prepared simultaneouslyor separately, are compared. In such embodiments, the methods facilitatequality control of glycoprotein preparation. Alternatively oradditionally, some such embodiments facilitate monitoring of progress ofa particular culture producing a glycoconj ugate of interest (e.g., whensamples are removed from the culture at different time points and areanalyzed and compared to one another). In any of these embodiments,features of the glycan analysis can be recorded, for example in aquality control record. As indicated above, in some embodiments, acomparison is with a historical record of a prior or standard batchand/or with a reference sample of glycoprotein.

In certain embodiments, the present disclosure may be utilized instudies to modify the glycosylation characteristics of a cell, forexample to establish a cell line and/or culture conditions with one ormore desirable glycosylation characteristics. Such a cell line and/orculture conditions can then be utilized, if desired, for production of aparticular target glycoconjugatc (e.g., glycoprotein) for which suchglycosylation characteristic(s) is/arc expected to be beneficial.

In certain embodiments, techniques of the present disclosure are appliedto glycans that are present on the surface of cells. In some suchembodiments, the analyzed glycans are substantially free ofnon-cell-surface glycans. In some such embodiments, the analyzedglycans, when present on the cell surface, are present in the context ofone or more cell surface glycoconjugates (e.g., glycoproteins orglycolipids).

In some particular embodiments, cell surface glycans are analyzed inorder to assess glycosylation of one or more target glycoproteins ofinterest, particularly where such target glycoproteins are not cellsurface glycoproteins. Such embodiments can allow one to monitorglycosylation of a target glycoprotein without isolating theglycoprotein itself. In certain embodiments, the present disclosureprovides methods of using cell-surface glycans as a readout of or proxyfor glycan structures on an expressed glycoprotein of interest. Incertain embodiments, such methods include, but are not limited to, postprocess, batch, screening or “in line” measurements of product quality.Such methods can provide for an independent measure of the glycosylationpattern of a produced glycoprotein of interest using a byproduct of theproduction reaction (e.g., the cells) without requiring the use ofdestruction of any produced glycoprotein. Furthermore, methods of thepresent disclosure can avoid the effort required for isolation ofproduct and the potential selection of product glycoforms that may occurduring isolation.

In certain embodiments, techniques of the present disclosure are appliedto glycans that are secreted from cells. In some such embodiments, theanalyzed glycans are produced by cells in the context of aglycoconjugate (e.g., a glycoprotein or glycolipid).

Techniques described herein can be used to detect desirable orundesirable glycans, for example to detect or quantify the presence ofone or more contaminants in a product, or to detect or quantify thepresence of one or more active or desired species.

In various embodiments the methods can be used to detect biomarkersindicative of, e.g., a disease state, prior to the appearance ofsymptoms and/or progression of the disease state to an untreatable orless treatable condition, by detecting one or more specific glycanswhose presence or level (whether absolute or relative) may be correlatedwith a particular disease state (including susceptibility to aparticular disease) and/or the change in the concentration of suchglycans over time.

In certain embodiments, the methods facilitate detection of glycans thatare present at very low levels in a source (e.g., a biological sample).In such embodiments, it is possible to separate over 10, 20, 30, 40, 50,60, 70, 80, 90 or 100 glycan components of a mixture, and to detect andoptionally quantify the levels of glycans comprising between 0.1% and5%, e.g., between 0.1% and 2%, e.g., between 0.1% and 1% of the originalglycan preparation. In certain embodiments, it is possible to detectand/or optionally quantify the levels of glycans at between about 0.1fmol to about 1 mmol.

In some embodiments, the techniques may be combined with one or moreother technologies for the detection, analysis, and or isolation ofglycans or glycoconjugates.

Thus, in certain embodiments, the methods comprise releasing N-linkedglycans from a glycoconjugate or cell surface to provide a glycanpreparation that includes a mixture of N-glycans. In certainembodiments, the mixture of N-glycans is provided via cleavage ofN-linked glycans from a glycoprotein after the cell surfaceglycoproteins have been liberated from the cell (e.g., through treatmentwith one or more proteases and/or glycosidases). In certain embodiments,the mixture of N-glycans is provided via cleavage of N-linked glycansfrom cell surface glycoproteins that have not been liberated from thecell. N-linked glycans may be released (e.g., separated, cleaved,hydrolyzed) using a variety of chemical or enzymatic methods; seegenerally, Kamerling, Pure Appl. Chem. (1994) 66:2235-2238; Kamerlingand Vliegnenthart, in: Clinical Biochemistry, Principles, Methods,Applications, Volume 1 (A. N. Lawson, ed), Walter De Gruyter, Berlin(1989) pp. 175-263; and Allen and Kisailus, eds., Glycoconguates, MarcelDekker Inc., New York, 1992.

Thus, in one aspect, a multi-dimensional chromatographic method for theseparation of a glycan preparation is provided which comprises the stepsof:

(i) cleaving N-linked glycans from a glycoprotein preparation to providea glycan preparation that includes a mixture of N-glycans, wherein atleast one of the N-glycans in the glycan preparation is negativelycharged; and

(ii) separating the glycan preparation by anion-exchange chromatographyand at least one secondary chromatographic technique.

Any of a variety of glycosidases that cleave glycan structures fromglycoproteins, or cell surface glycoproteins, may be used in accordancewith the present disclosure. Several examples of such glycosidases arereviewed in R. A. O'Neill, Enzymatic release of oligosaccharides fromglycoproteins for chromatographic and electrophoretic analysis, J.Chromatogr. A 720, 201-215. 1996; and S. Prime, et al., Oligosaccharidesequencing based on exo- and endo-glycosidase digestion and liquidchromatographic analysis of the products, J. Chromatogr. A 720, 263-274,1996. In certain embodiments, the enzyme PNGase F (Peptide N-GlycosidaseF) is used to remove glycans from a glycoprotein. PNGase F is an amidasethat cleaves the amide bond between the innermost GlcNAc and asparagineresidues of high mannose, hybrid, and complex oligosaccharides fromN-linked glycoproteins. Other suitable enzymes that can be used tocleave glycan structures from glycoproteins in accordance with thepresent disclosure include, but are not limited to, PNGase A andendoglycosidases (Endo-H). Those of ordinary skill in the art will beaware of other suitable enzymes for cleavage of glycans fromglycoproteins. In certain embodiments, a plurality of enzymes is used tocleave glycan structures from a glycoprotein.

To improve the accessibility of the glycosylation site to the enzyme,most glycoproteins require a protein denaturation step. Typically, thisis accomplished by using detergents and disulfide-reducing agents,although methods of denaturing a glycoprotein for use in accordance withthe present disclosure are not limited to the use of such agents. Forexample, exposure to high temperature can be sufficient to denature aglycoprotein such that a suitable enzyme for cleaving glycan structuresis able to access the cleavage site. In certain embodiments, acombination of detergents, disulfide-reducing agents, high temperature,and/or other agents or reaction conditions is employed to denature aglycoprotein. It is noted that glycans located at conserved Fc sites inimmunoglobulin G (IgG) are easily cleaved by PNGase F. Thus, a proteindenaturation step is typically not required for IgG molecules. PNGase Fis also capable of removing glycans in dilute ammonium hydroxidesolution. Thus, use of PNGase F to cleave glycans from glycoproteins hasthe advantage that the dilute ammonium hydroxide may additionally aid insolubility and some unfolding of the protein substrates.

Additionally, N-linked glycans may be cleaved from a glycoprotein usingchemical methods. For example, an N-linked glycan may be released viatreatment with hydrazine to provide a hydrazide of the N-glycan (i.e.,hydrazinolysis).

Additionally, following cleavage of the N-linked glycan from theglycoprotein or cell-surface glycoprotein, the N-glycans may be purifiedto remove non-carbohydrate contaminants, such as salts, chemicals, anddetergents used in enzymatic digests. The methods of purification mayinclude, but are not limited to, the use of C18 and graphitized carboncartridges and spin columns. In other embodiments, the method ofpurification may include a step of acetone precipitation ofproteinaceous material from an ice-cold aqueous solution containing bothproteins and glycans.

In certain embodiments, prior to separation according to the presentdisclosure, some or all of the N-glycans in the glycan preparation maybe derivatized with a label agent (e.g., a fluorescent or UV-activelabel). This label enables a higher sensitivity of detection of theglycan during chromatographic separation. Labeling agents for thispurpose are described in the art, e.g., see Anumula, Anal. Biochem.(2006) 350:1-23; Lamari et al., J. Chromatogr. B (2003) 793:15-36; Biggeet al., Anal. Biochem. (1995) 230:229-238, and references providedtherein.

Thus, in certain embodiments, a multi-dimensional chromatographic methodfor the separation of a mixture of N-glycans is provided which comprisesthe steps of:

(i) cleaving N-linked glycans from a glycoprotein preparation to providea glycan preparation that includes a mixture of N-glycans, wherein atleast one of the N-glycans in the glycan preparation is negativelycharged; and

(ii) reacting the glycan preparation with a labeling agent to provide aglycan preparation that includes a mixture of labeled N-glycans; andthen

(iii) separating the glycan preparation by anion-exchange chromatographyand at least one secondary chromatographic technique.

Exemplary fluorescent labeling agents include, but are not limited to,2-d aminobenzoic acid (2AA); 3-aminobenzoic acid (3AA); 4-aminobenzoicacid (4AA); anthranilic acid (AA); 2-aminopyridine (2AP);2-aminobenzamide (2AB); 3-aminobenzamide (3AB); 4-aminobenzamide (4AB);2-aminobenzoic ethyl etser (2ABEE); 3-aminobenzoic ethyl etser (3ABEE);4-aminobenzoic ethyl etser (4ABEE); 2-aminobenzonitrile (2ABN);3-aminobenzonitrile (3ABN); 4-aminobenzonitrile (4ABN);3-(acetylamino)-6-aminoacridin (AA-AC); 2-aminoacridonc (AMAC);methylanthranilatc (MA); 6-aminoquinolinc (6AQ);8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS);2-aminonaphthalene-1,3,6-trisulfonate (ANT);8-aminopyrene-1,3,6-trisulfonic acid (APTS); 7-aminomethyl-coumarin(AMC); 2-amino(6-amido-biotinyl)pyridine (BAP);9-fluorenylmethoxy-carbonyl-hydrazide (FMOC-hydrazide);3,5-dimethylanthranilic acid, and 2-amino-4,5-dimethoxy-benzoic acid.

The present disclosure contemplates use of any and all known “labelingagents” for labeling of N-glycans, as provided above and herein.

Additionally, the present disclosure contemplates use of any and all“labeling agents” for labeling of N-glycans, encompassed by the formulae(I) and (II), as depicted below,

wherein

R₁′ and R₁″ are each independently —H, —NH₂, —NHR₂, —CONH₂, —COOH,—COR₃, —COOR₄, —SO₃, —SO₁R₅ where n is 1 or 2, or a substituted orunsubstituted, cyclic or acyclic, branched or unbranched alkyl,substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkenyl, substituted or unsubstituted, cyclic or acyclic, branched orunbranched alkynyl, substituted or unsubstituted, cyclic or acyclic,branched or unbranched heteroalkyl, substituted or unsubstituted aryl orsubstituted or unsubstituted heteroaryl group, or when attached toadjacent carbon atoms R₁′ and R₁″ may be taken together with the atomsto which they are attached to form a 5- to 7-membered ring optionallycontaining a heteroatom selected from O, N or S;

R₂, R₃, R₄ and R₅ are each independently —H or substituted orunsubstituted, cyclic or acyclic, branched or unbranched alkyl,substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkenyl, substituted or unsubstituted, cyclic or acyclic, branched orunbranched alkynyl, substituted or unsubstituted, cyclic or acyclic,branched or unbranched heteroalkyl, substituted or unsubstituted aryl orsubstituted or unsubstituted heteroaryl group; R₆ is —H, NH₂, —NHR₂,—CONH₂, —COOH, —COR₃, —COOR₄, —SO₃ or —SO—R₅ where n is 1 or 2;

R₇′ and R₇″ arc each independently —H, —NH₂, —NHR₂, —CONH₂, —COOH,—COR₃, —COOR₄, —SO₃, —SO_(n)R₅ where n is 1 or 2, or an substituted orunsubstituted, cyclic or acyclic, branched or unbranched alkyl,substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkenyl, substituted or unsubstituted, cyclic or acyclic, branched orunbranched alkynyl, substituted or unsubstituted, cyclic or acyclic,branched or unbranched heteroalkyl, substituted or unsubstituted aryl orsubstituted or unsubstituted heteroaryl group, or when attached toadjacent carbon atoms R₁ and R₁′ may be taken together with the atoms towhich they are attached to form a 5- to 7-membered ring optionallycontaining a heteroatom selected from O, N or S, and

wherein any one of the hydrogen atoms is optionally isotopically labeledas ²H or ³H; any one of the carbon atoms is optionally isotopicallylabeled as ¹³C; any one of the oxygen atoms is optionally isotopicallylabeled as ¹⁸O; any one of the nitrogen atoms is optionally isotopicallylabeled as ¹⁵N; and any one of the sulfur atoms is optionallyisotopically labeled as ³³S or ³⁴S.

In certain embodiments, R₁′ and R₁″ are each independently —H, —NH₂,—NHR₂, —CONH₂, —COOH, —COR₃, —COOR₄, —SO₃, —SO_(n)R₅ where n is 1 or 2,or unsubstituted, cyclic or acyclic alkyl; unsubstituted, cyclic oracyclic alkenyl; unsubstituted, cyclic or acyclic alkynyl;unsubstituted, cyclic or acyclic heteroalkyl; unsubstituted aryl, orunsubstituted heteroaryl group, or when attached to adjacent carbonatoms R₁′ and R₁″ may be taken together with the atoms to which they areattached to form a 5- to 7-membered ring optionally containing aheteroatom selected from O, N or S.

In certain embodiments, R₂, R₃, R₄ and R₅ are each independently H orunsubstituted, cyclic or acyclic alkyl; unsubstituted, cyclic or acyclicalkenyl; unsubstituted, cyclic or acyclic alkynyl; unsubstituted, cyclicor acyclic heteroalkyl, unsubstituted aryl or unsubstituted heteroarylgroup.

In certain embodiments, R₇′ and R₇″ are each, independently, —H, —NH₂,—NHR₂, —CONH₂, —COOH, —COOR₄, —SO₃, —SO_(n)R₅ where n is 1 or 2, orunsubstituted, cyclic or acyclic alkyl, unsubstituted, cyclic or acyclicalkenyl, unsubstituted, cyclic or acyclic alkynyl, unsubstituted, cyclicor acyclic heteroalkyl, unsubstituted aryl or unsubstituted heteroarylgroup, or when attached to adjacent carbon atoms R₁ and R₁′ may be takentogether with the atoms to which they are attached to form a 5- to7-membered ring optionally containing a heteroatom selected from O, N orS.

The above labeling agents are used to label the glycan via reaction ofthe amine function group of the labeling agent with the N-glycan'sreducing (—CHO) end by reductive amination (see Scheme 1). One ofordinary skill in the art will appreciate that a wide variety ofreaction conditions may be employed to promote this reductive aminationreaction, therefore, a wide variety of reaction conditions areenvisioned; see generally, March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, M. B. Smith and J. March, 5^(th)Edition, John Wiley & Sons, 2001, and Comprehensive OrganicTransformations, R. C. Larock, 2^(nd) Edition, John Wiley & Sons, 1999.Suitable reductive amination conditions include providing a reducingagent, such as NaCNBH₃ or NaBH(OAc)₃, and maintaining an acidic toslightly acidic pH of the reaction mixture.

Labeling of the N-glycan is not limited to derivatization of thereducing end. The present disclosure also provides suitable labelingagents for tagging other functional groups present on the glycan moiety.For example, as depicted in Scheme 2, 1,2-diamino functionalizedlabeling agents, such as 1,2-diamino-4,5-methylenedioxy-benzene (DMB)and ortho-phenylenediamine (OPD), are suitable for tagging via reactionwith the alpha-keto acid functional group of sialic acids.

Furthermore, after labeling and separation steps according to themethods described herein, the isolated labeled N-glycans may be purifiedto remove non-carbohydrate contaminants, such as salts, excesschemicals, and acids, used during the labeling reaction.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this disclosure, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001;Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)Edition, Cambridge University Press, Cambridge, 1987.

In general, the term “substituted” refers to the replacement of hydrogenradicals in a given structure with the radical of a specifiedsubstituent. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. As used herein, the term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, any of the substituents described herein and any combinationthereof that results in the formation of a stable moiety. The presentdisclosure contemplates any and all such combinations in order to arriveat a stable substituent/moiety. For purposes of this disclosure,heteroatoms such as nitrogen may have hydrogen substituents and/or anysuitable substituent as described herein which satisfy the valencies ofthe heteroatoms and results in the formation of a stable moiety. Theterm “stable moiety,” as used herein, preferably refers to a moietywhich possess stability sufficient to allow manufacture, and whichmaintains its integrity for a sufficient period of time to be useful forthe purposes detailed herein.

The term “alkyl,” as used herein, refers to saturated, cyclic oracyclic, branched or unbranched, substituted or unsubstitutedhydrocarbon radicals derived from a hydrocarbon moiety containingbetween one and twenty carbon atoms by removal of a single hydrogenatom. In some embodiments, the alkyl group contains 1-20 carbon atoms.In another embodiment, the alkyl group employed contains 1-15 carbonatoms. In another embodiment, the alkyl group employed contains 1-10carbon atoms. In another embodiment, the alkyl group employed contains1-8 carbon atoms. In another embodiment, the alkyl group employedcontains 1-5 carbon atoms. Examples of alkyl radicals include, but arenot limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl,sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl,n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, andthe like, which may bear one or more sustitutents. Alkyl groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,cyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkyl, cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkenyl, cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkynyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedamino, substituted or unsubstituted hydroxy, substituted orunsubstituted thio, alkyloxy, aryloxy, alkyloxyalkyl, azido, oxo, cyano,halo, isocyano, nitro, nitroso, azo, —CONH₂, —COOH, —COR₃, —COOR₄, —SO₃,—SO_(n)R₅, wherein n is 1 or 2, and R₂, R₃, R₄ and R₅ are eachindependently —H or substituted or unsubstituted, cyclic or acyclic,branched or unbranched alkyl; haloalkyl, alkoxyalkyl, substituted orunsubstituted, cyclic or acyclic, branched or unbranched alkenyl;substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkynyl; substituted or unsubstituted, cyclic or acyclic, branched orunbranched cycloalkyl, substituted or unsubstituted, cyclic or acyclic,branched or unbranched cycloheteroalkyl, substituted or unsubstitutedaryl or substituted or unsubstituted heteroaryl group).

The term “cycloalkyl” refers to a cyclic alkyl group, as defined herein.Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclhexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like, which may bear one or moresustitutents. Cycloalkyl group substituents include, but are not limitedto, any of the substituents described herein, that result in theformation of a stable moiety (e.g., cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkyl, cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenyl, cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkynyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted amino, substituted orunsubstituted hydroxy, substituted or unsubstituted thio, haloalkyl,alkyloxy, aryloxy, alkyloxyalkyl, azido, cyano, halo, isocyano, nitro,nitroso, azo, oxo, —CONH₂, —COOH, —COR₃, —COOR₄, —SO₃, —SO_(n)R₅,wherein n is 1 or 2, and R₂, R₃, R₄ and R₅ are each independently —H orsubstituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkyl; substituted or unsubstituted, cyclic or acyclic, branched orunbranched alkoxyalkyl; substituted or unsubstituted, cyclic or acyclic,branched or unbranched alkenyl; substituted or unsubstituted, cyclic oracyclic, branched or unbranched alkynyl; substituted or unsubstituted,cyclic or acyclic, branched or unbranched cycloalkyl, substituted orunsubstituted, cyclic or acyclic, branched or unbranchedcycloheteroalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl group).

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a cyclic or acyclic, branched or unbranched, substituted orunsubstituted hydrocarbon moiety having at least one carbon-carbondouble bond by the removal of a single hydrogen atom. In certainembodiments, the alkenyl group contains 2-20 carbon atoms. In someembodiments, the alkenyl group contains 2-15 carbon atoms. In anotherembodiment, the alkenyl group employed contains 2-10 carbon atoms. Instill other embodiments, the alkenyl group contains 2-8 carbon atoms. Inyet another embodiments, the alkenyl group contains 2-5 carbons. Alkenylgroups include, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like, which may bear one or moresubstituents. Alkenyl group substituents include, but are not limitedto, any of the substituents described herein, that result in theformation of a stable moiety (e.g., cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkyl, cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenyl, cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkynyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted amino, substituted orunsubstituted hydroxy, substituted or unsubstituted thio, haloalkyl,haloalkyl, alkyloxy, aryloxy, alkyloxyalkyl, azido, cyano, halo,isocyano, nitro, nitroso, azo, oxo, —CONH₂, —COOH, —COR₃, —COOR₄, —SO₃,—SO_(n)R₅, wherein n is 1 or 2, and R₂, R₃, R₄ and R₅ are eachindependently —H or substituted or unsubstituted, cyclic or acyclic,branched or unbranched alkyl; substituted or unsubstituted, cyclic oracyclic, branched or unbranched alkoxyalkyl; substituted orunsubstituted, cyclic or acyclic, branched or unbranched alkenyl;substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkynyl; substituted or un substituted, cyclic or acyclic, branched orunbranched cycloalkyl, substituted or unsubstituted, cyclic or acyclic,branched or unbranched cycloheteroalkyl, substituted or unsubstitutedaryl or substituted or unsubstituted heteroaryl group).

The term “alkynyl,” as used herein, refers to a monovalent group derivedfrom a cyclic or acyclic, branched or unbranched, substituted orunsubstituted hydrocarbon having at least one carbon-carbon triple bondby the removal of a single hydrogen atom. In certain embodiments, thealkynyl group contains 2-20 carbon atoms. In some embodiments, thealkynyl group contains 2-15 carbon atoms. In another embodiment, thealkynyl group employed contains 2-10 carbon atoms. In still otherembodiments, the alkynyl group contains 2-8 carbon atoms. In still otherembodiments, the alkynyl group contains 2-5 carbon atoms. Representativealkynyl groups include, but are not limited to, ethynyl, 2-propynyl(propargyl), 1-propynyl, and the like, which may bear one or moresubstituents. Alkynyl group substituents include, but are not limitedto, any of the substituents described herein, that result in theformation of a stable moiety (e.g., cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkyl, cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenyl, cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkynyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted amino, substituted orunsubstituted hydroxy, substituted or unsubstituted thio, haloalkyl,alkyloxy, aryloxy, alkyloxyalkyl, azido, cyano, halo, isocyano, nitro,nitroso, azo, oxo, —CONH₂, —COOH, —COR₃, —COOR₄, —SO₃, —SO_(n)R₅,wherein n is 1 or 2, and R₂, R₃, R₄ and R₅ are each independently —H orsubstituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkyl; substituted or unsubstituted, cyclic or acyclic, branched orunbranched alkoxyalkyl; substituted or unsubstituted, cyclic or acyclic,branched or unbranched alkenyl; substituted or unsubstituted, cyclic oracyclic, branched or unbranched alkynyl; substituted or unsubstituted,cyclic or acyclic, branched or unbranched cycloalkyl, substituted orunsubstituted, cyclic or acyclic, branched or unbranchedcycloheteroalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl group).

The term “heteroalkyl,” as used herein, refers to an alkyl moiety, asdefined herein, which includes saturated, cyclic or acyclic, branched orunbranched, substituted or unsubstituted hydrocarbon radicals, whichcontain one or more oxygen, sulfur, nitrogen, phosphorus, or siliconatoms, e.g., in place of carbon atoms. In certain embodiments,hetereoalkyl moieties are substituted by independent replacement of oneor more of the hydrogen atoms thereon with one or more substituents.Heteroalkyl substituents include, but are not limited to, any of thesubstituents described herein, that result in the formation of a stablemoiety (e.g., cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkyl, cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkenyl, cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted amino, substituted or unsubstituted hydroxy,substituted or unsubstituted thio, alkyloxy, aryloxy, alkyloxyalkyl,azido, cyano, halo, isocyano, nitro, nitroso, azo, oxo, —CONH₂, —COOH,—COR₃, —COOR₄, —SO₃, —SO_(n)R₅, wherein n is 1 or 2, and R₂, R₃, R₄ andR₅ are each independently —H or substituted or unsubstituted, cyclic oracyclic, branched or unbranched alkyl; substituted or unsubstituted,cyclic or acyclic, branched or unbranched alkoxyalkyl; substituted orunsubstituted, cyclic or acyclic, branched or unbranched alkenyl;substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkynyl; substituted or unsubstituted, cyclic or acyclic, branched orunbranched cycloalkyl, substituted or unsubstituted, cyclic or acyclic,branched or unbranched cycloheteroalkyl, substituted or unsubstitutedaryl or substituted or unsubstituted heteroaryl group).

As used herein the term “haloalkyl” designates a C_(n)H_(2n+1) grouphaving from one to 2n+1 halogen atoms which may be the same ordifferent. Examples of haloalkyl groups include CF₃, CH₂C₁, C₂H₃BrCl,C₃H₅F₂, or the like. Similarly, the term haloalkoxy designates anOC_(n)H_(2n+1) group having from one to 2n+1 halogen atoms which may bethe same or different.

The term “alkoxyalkyl”, as used herein, refers to an alkyl group ashereinbefore defined substituted with at least one alkyloxy group.

The term “cycloheteroalkyl,” as used herein, refers to a cyclicheteroalkyl group as defined herein. A cycloheteroalkyl group refers toa fully saturated 3- to 10-membered ring system, which includes singlerings of 3 to 8 atoms in size. These cycloheteroalkyl rings includethose having from one to three heteroatoms independently selected fromoxygen, sulfur, and nitrogen, in which the nitrogen and sulfurheteroatoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. In certain embodiments, the termcycloheteroalkyl refers to a 5-, 6-, or 7-membered ring or polycyclicgroup wherein at least one ring atom is a heteroatom selected from O, S,and N (wherein the nitrogen and sulfur heteroatoms may be optionallyoxidized), and the remaining ring atoms are carbon, the radical beingjoined to the rest of the molecule via any of the ring atoms. Examplesof cycloheteroalkyl ring systems included in the term as designatedherein are the following rings wherein X₁ is NR′, O or S, and R′ is H oran optional substituent as defined herein:

Exemplary cycloheteroalkyls include azacyclopropanyl, azacyclobutanyl,1,3-diazatidinyl, piperidinyl, piperazinyl, azocanyl, thiaranyl,thietanyl, tetrahydrothiophenyl, dithiolanyl, thiacyclohexanyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropuranyl, dioxanyl,oxathiolanyl, morpholinyl, thioxanyl, tetrahydronaphthyl, and the like,which may bear one or more substituents. Substituents include, but arenot limited to, any of the substituents described herein, that result inthe formation of a stable moiety (e.g., cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkyl, cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenyl, cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkynyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted amino, substituted orunsubstituted hydroxy, substituted or unsubstituted thio, haloalkyl,alkyloxy, aryloxy, alkyloxyalkyl, azido, cyano, halo, isocyano, nitro,nitroso, azo, oxo, —CONH₂, —COOH, —COR₃, —COOR₄, —SO₃, —SO_(n)R₅,wherein n is 1 or 2, and R₂, R₃, R₄ and R₅ are each independently —H orsubstituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkyl; substituted or unsubstituted, cyclic or acyclic, branched orunbranched alkoxyalkyl; substituted or unsubstituted, cyclic or acyclic,branched or unbranched alkenyl; substituted or unsubstituted, cyclic oracyclic, branched or unbranched alkynyl; substituted or unsubstituted,cyclic or acyclic, branched or unbranched cycloalkyl, substituted orunsubstituted, cyclic or acyclic, branched or unbranchedcycloheteroalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl group)

The term “aryl,” as used herein, refer to stable aromatic mono- orpolycyclic ring system having 3-20 ring atoms, of which all the ringatoms are carbon, and which may be substituted or unsubstituted. Incertain embodiments of the present disclosure, “aryl” refers to a mono,bi, or tricyclic C₄-C₂₀ aromatic ring system having one, two, or threearomatic rings which include, but not limited to, phenyl, biphenyl,naphthyl, and the like, which may bear one or more substituents. Arylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,cyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkyl, cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkenyl, cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkynyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedamino, substituted or unsubstituted hydroxy, substituted orunsubstituted thio, haloalkyl, alkyloxy, aryloxy, alkyloxyalkyl, azido,cyano, halo, isocyano, nitro, nitroso, azo, —CONH₂, —COOH, —COR₃,—COOR₄, —SO₃, —SO_(n)R₅, wherein n is 1 or 2, and R₂, R₃, R₄ and R₅ areeach independently —H or substituted or unsubstituted, cyclic oracyclic, branched or unbranched alkyl; substituted or unsubstituted,cyclic or acyclic, branched or unbranched alkoxyalkyl; substituted orunsubstituted, cyclic or acyclic, branched or unbranched alkenyl;substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkynyl; substituted or unsubstituted, cyclic or acyclic, branched orunbranched cycloalkyl, substituted or unsubstituted, cyclic or acyclic,branched or unbranched cycloheteroalkyl, substituted or unsubstitutedaryl or substituted or unsubstituted heteroaryl group).

The term “heteroaryl,” as used herein, refer to stable aromatic mono- orpolycyclic ring system having 3-20 ring atoms, of which one ring atom isselected from S, O, and N; zero, one, or two ring atoms are additionalheteroatoms independently selected from S, O, and N; and the remainingring atoms are carbon, the radical being joined to the rest of themolecule via any of the ring atoms. Exemplary heteroaryls include, butare not limited to pyrrolyl, pyrazolyl, imidazolyl, pyridinyl,pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl,pyyrolizinyl, indolyl, quinolinyl, isoquinolinyl, benzoimidazolyl,indazolyl, quinolinyl, isoquinolinyl, quinolizinyl, cinnolinyl,quinazolynyl, phthalazinyl, naphthridinyl, quinoxalinyl, thiophenyl,thianaphthenyl, furanyl, benzofuranyl, benzothiazolyl, thiazolynyl,isothiazolyl, thiadiazolynyl, oxazolyl, isoxazolyl, oxadiaziolyl,oxadiaziolyl, and the like, which may bear one or more substituents.Heteroaryl substituents include, but are not limited to, any of thesubstituents described herein, that result in the formation of a stablemoiety (e.g., cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkyl, cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkenyl, cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted amino, substituted or unsubstituted hydroxy,substituted or unsubstituted thio, haloalkyl, alkyloxy, aryloxy,alkyloxyalkyl, azido, cyano, halo, isocyano, nitro, nitroso, azo,—CONH₂, —COOH, —COR₃, —COOR₄, —SO₃, —SO_(n)R₅, wherein n is 1 or 2, andR₂, R₃, R₄ and R₅ are each independently —H or substituted orunsubstituted, cyclic or acyclic, branched or unbranched alkyl;substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkoxyalkyl; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched alkenyl; substituted or unsubstituted, cyclic or acyclic,branched or unbranched alkynyl; substituted or unsubstituted, cyclic oracyclic, branched or unbranched cycloalkyl, substituted orunsubstituted, cyclic or acyclic, branched or unbranchedcycloheteroalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl group)

The term “amino,” as used herein, refers to a group of the formula(—NH₂). A “substituted amino” refers either to a mono-substituted amine(—NHR^(h)) or a di-substituted amine (—NR^(h) ₂), wherein the R^(h)substituent is any substitutent as described herein that results in theformation of a stable moiety (e.g., cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkyl, cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenyl, cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkynyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted amino, substituted orunsubstituted hydroxy, haloalkyl, alkyloxy, aryloxy, alkyloxyalkyl,azido, cyano, halo, oxo, —CONH₂, —COOH, —COR₃, —COOR₄, —SO₃, —SO_(n)R₅wherein n is 1 or 2, and R₂, R₃, R₄ and R₅ are each independently —H orsubstituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkyl; substituted or unsubstituted, cyclic or acyclic, branched orunbranched alkoxyalkyl; substituted or unsubstituted, cyclic or acyclic,branched or unbranched alkenyl; substituted or unsubstituted, cyclic oracyclic, branched or unbranched alkynyl; substituted or unsubstituted,cyclic or acyclic, branched or unbranched cycloalkyl, substituted orunsubstituted, cyclic or acyclic, branched or unbranchedcycloheteroalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl group). In certain embodiments, the Rhsubstituents of the di-substituted amino group(—NR^(h) ₂) form a 5- to6-membered cycloheteroalkyl ring.

The term “hydroxy,” or “hydroxyl,” as used herein, refers to a group ofthe formula (—OH). A “substituted hydroxyl” refers to a group of theformula (—OR^(i)), wherein R^(i) can be any substitutent which resultsin a stable moiety (e.g., cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkyl, cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkenyl, cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkynyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, —CONH₂, —COOH, —COR₃, —COOR₄, —SO₃, —SO_(n)R₅, wherein n is1 or 2, and R₂, R₃, R₄ and R₅ are each independently —H or substitutedor unsubstituted, cyclic or acyclic, branched or unbranched alkyl;substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkoxyalkyl; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched alkenyl; substituted or unsubstituted, cyclic or acyclic,branched or unbranched alkynyl; substituted or unsubstituted, cyclic oracyclic, branched or unbranched cycloalkyl, substituted orunsubstituted, cyclic or acyclic, branched or unbranchedcycloheteroalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl group).

The term “thio” or “thiol” as used herein, refers to a group of theformula (—SH). A “substituted thiol” refers to a group of the formula(—SR^(r)), wherein R^(r) can be any substitutent which results in astable moiety (e.g., cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkyl, cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkenyl, cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkynyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, —CONH₂, —COOH, —COR₃, —COOR₄, —SO₃, —SO_(n)R₅, wherein n is1 or 2, and R₂, R₃, R₄ and R₅ are each independently —H or substitutedor unsubstituted, cyclic or acyclic, branched or unbranched alkyl;substituted or unsubstituted, cyclic or acyclic, branched or unbranchedalkoxyalkyl; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched alkenyl; substituted or unsubstituted, cyclic or acyclic,branched or unbranched alkynyl; substituted or unsubstituted, cyclic oracyclic, branched or unbranched cycloalkyl, substituted orunsubstituted, cyclic or acyclic, branched or unbranchedcycloheteroalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl group).

The term “alkyloxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted alkyl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule. The term “alkylthioxy” refers to a “substituted thiol” of theformula (—SR^(r)), wherein R^(r) is an optionally substituted alkylgroup, as defined herein, and the sulfur moiety is directly attached tothe parent molecule. The term “alkylamino” refers to a “substitutedamino” of the formula (—NR^(h) ₂), wherein R^(h) is, independently, ahydrogen or an optionally substituted alkyl group, as defined herein,and the nitrogen moiety is directly attached to the parent molecule.

The term “aryloxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted aryl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule. The term “arylamino,” refers to a “substituted amino” of theformula (—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted aryl group, as defined herein, and the nitrogenmoiety is directly attached to the parent molecule. The term“arylthioxy” refers to a “substituted thiol” of the formula (—SR^(r)),wherein R^(r) is an optionally substituted aryl group, as definedherein, and the sulfur moiety is directly attached to the parentmolecule.

The term “alkyloxyalkyl” or “alkoxyalkyl” as used herein refers to analkyloxy group, as defined herein, attached to an alkyl group attachedto the parent molecule.

The term “azido,” as used herein, refers to a group of the formula(—N₃).

The term “cyano,” as used herein, refers to a group of the formula(—CN).

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo,—Br), and iodine (iodo, —I).

The term “isocyano,” as used herein, refers to a group of the formula(—NC).

The term “nitro,” as used herein, refers to a group of the formula(—NO₂).

The term “nitroso,” as used herein, refers to a group of the formula(—N═O).

The term “azo,” as used herein, refers to a group of the formula (—N₂).

The term “oxo,” as used herein, refers to a group of the formula (═O).

In some embodiments, the techniques may be combined with one or moreother technologies for the detection, analysis, and or isolation ofglycans or glycoconjugates. It will be appreciated that once theN-glycans have been separated according to the methods described hereinthey may be further analyzed by any technique. For example, theN-glycans may be analyzed by mass spectrometry or nuclear magneticresonance (e.g., using TOCSY, NOESY or HSQC type experiments, etc. todetermine structural features). Mass spectroscopic analysis can beperformed using methods such as ESI-MS, ESI-MS/MS, MALDI-TOF-MS,MALDI-TOF/TOF-MS, tandem MS, etc.

The methods will be more specifically illustrated with reference to thefollowing examples. However, it should be understood that the methodsare not limited by these examples in any manner.

EXAMPLES Example 1

N-glycans contain different number of sialic acids. In one experiment,we therefore chose to perform the first dimensional separation based onthe number of charges of the glycans using anion-exchange chromatography(AIEC). After AIEC, each individual fraction was further separated,using an secondary chromatographic technique. In this particular examplewe used (a) reverse-phase chromatography to further separate the AIECglycan fraction corresponding to neutral glycans and/or (b) normal-phaseamide chromatography to further separate the AIEC glycan fractionscorresponding to acidic, negatively-charged glycans. In addition, theN-glycans were labeled with the fluorescent label 2-AB in order tofacilitate the sensitive and quantitative detection of the N-glycansduring the separation process.

AIEC separation of an N-glycan pool was performed on a DEAE column(TSK-gel, 7.5 mm×7.5 cm, Tosoh Inc.) using the following conditions:

-   -   Column temperature: 40° C.    -   Buffer A: 10% acetonitrile, 90% H₂O; pH 7.    -   Buffer B: 50% 500 mM ammonium acetate, 40% H₂O, 10%        acetonitrile; pH 7.

Gradient between Buffer A and Buffer B:

Time (min) Flow (ml/min) % A % B 0 0.5 100 0 5 0.5 100 0 95 0.5 40 60100 0.5 40 60 101 0.5 0 100 106 0.5 0 100 107 0.5 100 0 120 0.5 100 0121 0 100 0

FIG. 1 is a representative chromatogram of the AIEC separation usingthese conditions. The data indicate that the 2-AB labeled N-glycans wereseparated into several groups based on the number of charges theycarried.

After the first dimension of separation, fractions from AIEC werecollected offline and concentrated before being subjected to secondarychromatographic separations. Several fractions of 2AB-labeled glycanswere collected from the A1EC separation. Each fraction was lyophilizedand reconstituted into an appropriate volume for offline injection ontoa second secondary dimension of separation.

In one embodiment, the first fraction is further separated in a seconddimension by reverse-phase C18 HPLC separation. Separated peaks on thisC18 HPLC may be analyzed by offline MALDI MS or by online LC-MS.

In another embodiment, the first fraction is subjected to a seconddimension of separation using a normal-phase amide HPLC column.Separated peaks on this amide HPLC may be analyzed by offline MALDI MSor by online LC-MS. For example, the results of separating all of theAIEC fractions on a normal-phase amide HPLC column using an ammoniumformate buffer gradient of 0 to 50 mM ammonium formate are shown in FIG.4. In another embodiment, the later-eluting AIEC glycans (e.g., some orall of fractions 7-11) are subjected to a second offline dimension ofseparation using a graphitized carbon column HPLC method, instead of anormal-phase HPLC method (e.g., see Example 2). In another embodiment,some or all of the AIEC fractions are analyzed by a second dimension ofnormal-phase amide chromatography, and then by a third dimension ofchromatography, such as (but not limited to) graphitized carbon columnchromatography (e.g., see Example 2). In this embodiment, the glycanstructures may be analyzed by offline MALDI-MS or by online LC-MS.

Example 2

An N-glycan pool was obtained from a test glycoprotein, and the glycanswere fluorescently labeled. They were then fractionated viaanion-exchange chromatography, generating fractions “IEX fraction 1, 2,3”, etc. As described below, 10 of these fractions were then furtherseparated by either amide chromatography or PGC (porous graphitizedcarbon) chromatography. Without limitation, suitable amide columnsinclude the GlycoSep-N, Ludgersep-N1, or Tsk-gel amide-80 columns whileother suitable PGC columns include the Supelco envicarb and ThermoHypercarb columns.

In this experiment, certain fractions obtained from anion exchangechromatography (AIEX or IEX) were separated using an amide column in thesecond dimension (see FIGS. 5A-E and 6), while other fractions wereseparated using a PGC column in the second dimension (see FIGS. 5F-G and7). The areas of resolved peaks can be used to quantify the glycanspecies. Relative quantities can be obtained using peak ratios. Byspiking samples with a known amount of a fluorescently-labeledN,N′-diacetyl chitobiose standard absolute quantitation can be achieved.

A comparison of the panels within FIG. 4 (see Example 1) shows that thelater IEX fractions 7-9 and 11 (see FIGS. 4F-J) are in general less wellseparated on an amide column, than are the earlier IEX fractions 1-6 and10 (See FIGS. 4A-E, I). We observed a significant improvement in theseparation for fraction 7 using the PGC method for the second dimensionof separation (compare FIG. 5E and FIGS. 5F-G). Similar results wereobtained with fraction 8 (see below) and fraction 9 (data not shown).

The following are typical separation conditions that could be used foran amide column such as the GlycoSep-N column (4.6×250 mm, Prozyme) usedin this example:

-   -   Column Temperature: 15-50° C.    -   Buffer A: acetonitrile (50-100%).    -   Buffer B: 5-250 mM ammonium acetate (or formate or carbonate),        pH 4-8.

For example, the separations in FIGS. 5A and 5C were obtained using aGlycoSep-N column (4.6×250 mm, Prozyme) at 45° C., the following binarysolvent system (Buffer A: acetonitrile and Buffer B: 50 mM ammoniumacetate, pH 7), a 0.75 ml/min flow rate and the following elutiongradient:

Time (mins) % Buffer B 5 35 77 53 78 100 93 100 94 35 110 35

The separations in FIGS. 5B and 5D were obtained using the same columnsystem but a shallower 30-48% elution gradient. By tuning the gradientwe obtained better separation of fractions 3 and 5, than the steeper35-53% gradient described in the previous table.

The following are typical separation conditions that could be used for aPGC column such as the Hypercarb column (4.6×150 mm, Thermo) used inthis example:

-   -   Column Temperature: 15-40° C.    -   Buffer A: 2-100 mM ammonium acetate (or formate or carbonate),        pH 4.5-8.5.    -   Buffer B: 2-100 mM ammonium acetate (or formate or carbonate),        pH 4.5-8.5 in 20-60% acetonitrile.

For example, the separations in FIGS. 5F and 5G were obtained using aHypercarb column (4.6×150 mm, Thermo) at 30° C., the following binarysolvent system (Buffer A: 50 mM ammonium acetate, pH 7 and Buffer B:50:50 mixture of acetonitrile and 50 mM ammonium acetate, pH 7), a 0.6ml/min flow rate and the following elution gradient:

Time % Buffer B Time % Buffer B (mins) FIG. 5F (mins) FIG. 5G 5 42 5 4750 80 45 52 65 80 70 58 66 42 71 80 80 42 80 80

The separations in FIG. 7 were obtained using the same column and buffersystem but a different set of elution gradients:

% Buffer B Time (mins) FIG. 7A FIG. 7B FIG. 7C 0 38 43 44 5 38 43 44 6558 55 51 66 80 80 80 86 80 80 80 87 38 43 44 100 38 43 44

FIGS. 6 and 7 show that the chromatographic resolution of fraction 4 byan amide column (FIG. 6) was comparable to that attainable by a PGCcolumn (FIG. 7). FIGS. 8 and 9 compare the amide and PGC chromatogramsfor fraction 8 and show that the PGC method gave surprisingly betterseparation than the amide. In particular, the PGC method was able toseparate glycans which were not resolvable by the amide method. Similarresults were obtained for fractions 7 (see above) and 9 (data notshown).

EQUIVALENTS

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way.

While the methods have been described in conjunction with variousembodiments and examples, it is not intended that the methods be limitedto such embodiments or examples. On the contrary, the present disclosureencompasses various alternatives, modifications, and equivalents, aswill be appreciated by those of skill in the art.

While the methods have been particularly shown and described withreference to specific illustrative embodiments, it should be understoodthat various changes in form and detail may be made without departingfrom the spirit and scope of the present disclosure. Therefore, allembodiments that come within the scope and spirit of the presentdisclosure, and equivalents thereto, are intended to be claimed. Theclaims, descriptions and diagrams of the methods, systems, and assays ofthe present disclosure should not be read as limited to the describedorder of elements unless stated to that effect.

1-20. (canceled)
 21. A method of monitoring production of aglycoprotein, the method comprising: producing a glycoprotein in a cellculture; removing at least a first and second sample of theglycoprotein, wherein the samples include at least one charged N-glycan;adding a known quantity of a reference N-glycan to the samples, whereinthe reference N-glycan is labeled with a labeling agent; for each of thefirst and second samples: (i) separating the sample by anion-exchangechromatography to generate a plurality of fractions; (ii) separating afirst portion of the plurality of fractions by at least one secondarychromatographic technique to obtain first separated fractions; (iii)separating a second portion of the plurality of fractions by at leastone secondary chromatographic technique that differs from the secondarychromatographic technique from (ii) to obtain second separatedfractions; (iv) quantifying at least one N-glycan relative to thereference N-glycan in at least a portion of the first separatedfractions, or in at least a portion of the second separated fractions,or both; and comparing the result of (iv) from the first sample withthat for the second sample.
 22. The method of claim 21, wherein the atleast one secondary chromatographic technique from (ii) is selected fromthe group consisting of reversed phase liquid chromatography (RP),normal phase liquid chromatography (NP), ion-pairing reverse phasechromatography (IP-RP), size exclusion chromatography, affinitychromatography (AC), capillary electrophoresis (CE);fluorophore-assisted carbohydrate electrophoresis (FACE);electrochromatography, and micellar electrokinetic chromatography(MEKC).
 23. The method of claim 21, wherein the at least one secondarychromatographic technique from (ii) is a reversed phase chromatographictechnique.
 24. The method of claim 23, wherein the reversed-phasechromatographic technique employs a C18 reverse phase resin.
 25. Themethod of claim 23, wherein the reversed-phase chromatographic techniqueemploys a porous graphitized-carbon (PGC) resin.
 26. The method of claim21, wherein the at least one secondary chromatographic technique from(ii) is a normal phase chromatographic technique.
 27. The method ofclaim 26, wherein the normal phase chromatographic technique employs amodified silica gel.
 28. The method of claim 27, wherein the modifiedsilica gel is selected from the group consisting of cyano-modifiedsilica, amine-modified silica, and amide-modified silica.
 29. The methodof claim 21, wherein the at least one secondary chromatographictechnique from (iii) is selected from the group consisting of reversedphase liquid chromatography (RP), normal phase liquid chromatography(NP), ion-pairing reverse phase chromatography (IP-RP), size exclusionchromatography, affinity chromatography (AC), capillary electrophoresis(CE); fluorophore-assisted carbohydrate electrophoresis (FACE);electrochromatography, and micellar electrokinetic chromatography(MEKC).
 30. The method of claim 21, wherein the at least one secondarychromatographic technique from (iii) is a reversed phase chromatographictechnique.
 31. The method of claim 30, wherein the reversed-phasechromatographic technique employs a resin selected from the groupconsisting of a C18 reverse phase resin and a porous graphitized-carbon(PGC) resin.
 32. The method of claim 21, wherein the at least onesecondary chromatographic technique from (iii) is a normal phasechromatographic technique.
 33. The method of claim 32, wherein thenormal phase chromatographic technique employs a modified silica gel.34. The method of claim 33, wherein the modified silica gel is selectedfrom the group consisting of cyano-modified silica, amine-modifiedsilica, and amide-modified silica.
 35. The method of claim 21, whereinthe at least one secondary chromatographic technique from (ii) is anormal phase chromatographic technique and the at least one secondarychromatographic technique from (iii) is a reverse phase chromatographictechnique.
 36. The method of claim 21, wherein the first portion of theplurality of fractions comprises the first half to two-thirds of thefractions from the anion exchange chromatography.
 37. The method ofclaim 21, wherein the second portion of the plurality of fractionscomprises the second half to one-third of the fractions from the anionexchange chromatography.
 38. The method of claim 21, wherein theglycoprotein is a therapeutic glycoprotein.
 39. The method of claim 38,wherein the therapeutic glycoprotein is an antibody.
 40. The method ofclaim 39, wherein the antibody is a preparation of alemtuzumab,etanercept, adalimumab, abatacept, infliximab, bevacizumab, rituximab,natalizumab, or cetuximab.